CONTRIBUTION OF DEFECTIVE DNA REPAIR IN CANCER DEVELOPMENT AND TREATMENT

DNA is the most important component of a cell to maintain its integrity. The process of replication and transcription has to be accurate to stabilize the genome. However, Without the enzyme polymerase (with both exonuclease and proofreading activity) error rate in DNA replication is found to be 10-6 to 10-8 (Thomas A Kunkel, 2004). Along with the enzyme polymerase, there are other types of machinery to repair damaged DNA during replication and other biochemical activities. The bacterial transforming DNA was inactivated by creating a lesion in presence of UV light of lower wavelength. Incorporation of the enzyme-like agent from the yeast on photoactivation restored the activity of bacterial DNA (Claud S Rupert, 1962). The experimental result was very much evident and suggested the implication of a machinery for DNA repair. UV radiations are known to cause thymidine dimers; cellular uptake of acridine dye is inversely proportional to thymidine dimers present in a cell. Three cell lines (desquamated buccal cells, cells from asymptotic smokes and cancer cells from oral cavity) were exposed to UV radiation and treated with enzyme-like complex from yeast in a medium containing acridine. Cancer cells distinctly expressed the deficit of repair response for damaged DNA and were analyzed by least cellular uptake of acridine (Daniel Roth, 1969). This was a landmark experiment to describe the role of defective DNA repair in cancerization.
Both external and internal factors can result in DNA damage. Several signalling pathways come into the picture as a response to the damaged DNA. The outcome of signalling pathways decide the fate of a cell and is categorised into three sections, namely; DNA repair, damage tolerance, and apoptosis. Several mutagenic responses are directly linked to defect in DNA repair machinery and mutations in sensors which recognizes DNA damage (Wynand P Roos, 2016).

Significant DNA damages like single strand break (SSB), double-strand break (DSB) and stalled replication fork activates the damage response elements. Functioning of DDR cascade comprises of sensory molecules like DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 Related (ATR) and PI3K related kinases (PIKK). Mutations in ATM or ATR cannot activate the
tumour suppressing protein, p53 and apoptotic initiator protein, Caspase 2; as a result, cell proliferation continues without the checkpoint barriers in the cell cycle (Figure 2). Laps of DDR elements explains the intricacies leading to uncontrolled cell proliferation (Wynand P Roos, 2016). Apart from sensors which detect DNA damage, repair system itself would be a deficit to rectify the damaged DNA.
Several examples are quoted to explain the cause of cancer on the basis of a defect in DDR or defect in DNA repair.

Xeroderma Pigmentosum (XP):
Person having XP explicates a 1000 fold more chances to get skin cancer (Cleaver, 1969). The cells from XP patient showed that there was a defect in repairing the DNA damage caused by UV radiation. Subsequent investigation of the cells from XP patient showed the mutation in the component of Nucleotide Excision Repair (NER) system which blocked repair mechanism (Lehman, 2011). Due to the defective repair system the dimers (thymidine dimer) formed by the UV light cannot be eliminated; resulting in the specific skin cancer predisposition in XP patients.

Lynch Syndrome or Hereditary Non-Polyposis Colorectal Cancer (HNPCC):
Instability of repeats of microsatellite causes a familial pattern of colorectal cancer characterized by mutations in the homologues of mismatch repair proteins, MutS and MutL (Leach, 1993). Lynch syndrome significantly increased the risk of other cancer types like endometrial cancer, ovarian cancer, pancreatic cancer, kidney cancer, urinary tract cancer (Cancer.net, 2014).

Werner Syndrome (WS):
WRN is a protein known to have both exonuclease and helicase activity. WRN protein is a key protein in DSB repair by Non-Homologous end joining (NHEJ) or homologous recombination (HR). Mutations in WNR gene can cause predisposition to cancer and early aging (Gray, M. D., 1997).

Fanconi Anemia (FA):
FA pathways includes three complex protein systems involved in DNA repair mechanism. HR, NER, and mutagenic translesion synthesis are dependent on FA pathway which employs nuclear protein complexes to ubiquitinate FANCD2 and FANCI, which in turn results in the formation of repair complex. The absence of ubiquitination of FANCD2 and FANCI unambiguously leads to collapse in the repair system causes uncontrolled cell proliferation (Moldovan, G.L, 2009).

Breast and Ovarian cancer:
BRCA1 is a vital component involved in DNA repair mechanism and is found to be in association with RAD51, protein functions in DSB repair system by homologous recombination. Mutant BRCA1 evidently altered homologous and non-homologous DNA integration and DSB repair. Maintenance of genomic integrity is chiefly regulated by BRCA1; thus, mutation of which results in the predisposition to cancer (Moynahan, M. E, 1999).

These instances evidently explain the consequences of faulty DNA damage repair. However, DNA repair mechanism itself act as a barrier in therapeutics of many cancer types. Anti-cancer agents target the DNA and create breaks in them so as to draw the cancer cells to apoptotic pathway. Cancer caused by other means (with unaltered repair machinery) would bring out efficient repair system to neutralize the activity of the anti-cancer drug. In such cases, it is obvious to use the supportive drug to inactivate the repair process. Functioning of supportive drugs is based on the pattern of damage caused by the initial drug used to damage the DNA. Thus it is a big challenge to reduce the risk that would be caused by the usage of the secondary drug molecule. This strategy cannot be employed to cancer caused by faulty DNA damage repair system. Cancer caused by defective DNA repair must be treated with a drug molecule which could either create cell senescence or cell death (apoptosis, necrosis or autophagy).
Any repair mechanism could be specifically targeted to get a better result for a cancer type with DNA damaging agent.

Targeting BER:
Anti-cancerous alkylating agents results in alkylated and oxidized bases which can be removed by Base excision repair mechanism. Apurinic or Apyrimidinic endonucleases play a vital role in BER. Inhibition of APE1 blocks the BER pathway and thereby alkylating agent could damage the DNA molecules go arrest the cell cycle. AP site can also be targeted to modify it by the virtue of which APE1 fails to bind to it. Methoxyamine (MX) is a tiny molecule with lesser Km value to APE1. Hence MX strongly binds with AP site and blocks APE1 to proceed with the repair process. APE1 is a multifunctional enzyme and is better to target APE1 to block all its functions. Thus, the combination of Temozolomide (Alkylating agent), E3330 (blocks redox activity of APE1) and MX can increase the efficiency of therapeutic activity and promising results are obtained.

Targeting HR and NHEJ:
Exposure to IR for a prolonged duration, ROS is a highly potential agent to create DSB. Meanwhile, these therapeutics cannot be efficient due to DDR. DDR will bring out the activation of HR or NHEJ repair systems which neutralizes the therapeutic activity of IR and ROS. DDR is a function mediated by ATM, ATR, and DNA-PK which transduces the signals to activate repair pathway. Inhibition of ATM and ATR were not significance due to the side effects and sensitivity to switching over to other cancer types (Collis SJ, 2005). Thus targeting DNA-PK looks promising to increases the therapeutic activity with fewer side effects. DNA-PK inhibitors like vanillin, NU7026 are very effective and efficiently blocked the repair system. Some of the
DNA damaging agents can even inhibit DNA-PK thereby performs the dual function and reduces the risk of side effect. For instance, inhibitors of Topoisomerase 2 even blocks NHEJ. Highly advanced and accurate technique is the use of siRNA and it is found to reduce the activity of ATM, ATR and DNA-PK by 90% and was significantly higher than wortmannin (Collis SJ, 2003).

Targeting direct repair:
O6 Methyl Guanine-DNA-Methyl Transferase (MGMT) results in resistance to many anticancer drugs, like Temozolomide, dacarbazine, BCNU by transferring the adduct to its own cysteine residue. Thus MGMT gene is targeted and subjected for methylation of its gene. Analogue of guanine is used to suppress the enzymatic activity of MGMT (Yongjian Zhu, 2009).
Therapeutic actions are very crucial and have to be checked for side effects and other possible consequences. Personalised medicine would possibly give a better result; along with which whole genome sequencing and exome sequencing techniques should be employed for cancer therapy. Each cancer patterns have to be sequestered with genome sequencing through which medication could be done at the molecular level. Cancer with a family history should be treated with suitable prognostic methods.





References:
1. Thomas A Kunkel, JBC – Apr 23, 2004
2. Claud S Rupert, The journal of General Physiology, Vol 45,1962
3. Daniel Roth and Harold H Sage, Cancer, Sept 1969.
4. Cleaver, J. E. Proc. Natl Acad. Sci. USA 63, 1969
5. Lehmann, A. R., McGibbon, D. & Stefanini, 6, 70 (2011).
6. Leach, F. S. et al. Cell 75, (1993)
7. Cancer.net, Editorial board, 12/2014
8. Gray, M. D. et al. Nat. Genet. 17, (1997).
9. Moldovan, G. L. & D’Andrea, A. D. Annu. Rev. Genet. 43, (2009).
10. Moynahan, M. E., Chiu, J. W., Koller, B. H. & Jasin, M. 1999
11. Yongjian Zhu a,d,e, Jue Hu b,d,f, Yiduo Hu c,g, Weiguo Liu a
12. Collis SJ, DeWeese TL, Jeggo PA, Parker AR. Oncogene 2005.

Diagrams are available and could be requested through email
The article is written by using the data from several research articles and are subjected for copyright. Hence, they are referenced accordingly.

CANCER AND ITS OTHER PHASES/FACES

THERE COULD BE A REASON FOR CANCER WHICH IS WAY BEYOND IMAGINATION...!!!

Cancer is one of the key reasons for death all over the world. Several schools of scientists opined that cancer is because mainly due to tobacco consumption, alcoholism, unscientific food habit, exposure to radiation and even due to heredity. I do agree with all these key reasons; however they may not be the key reason in many of the cases. One such instance I had come across, a 95 years old person remained without any health issues and he was addicted for consumption of raw tobacco leaves from the age of 13…! So, there’s some other reason which triggers the onset of cancer. If the cigarettes are known to cause cancer, then half of the world’s population should be of cancer patients. In traditional country like India, several village people are used to have beetle leaves after lunch/dinner as a digestive stimulant. Still, they are the healthiest compared well civilized urban people. Even in this case modern science fails to answer to these exceptions. In north India, a kind of people called NAGAs are very much addicted to use chillum filled with marijuana. Mysterious thing about them is, there are many NAGAs whose age is still big question and is unpredictable. Does the reason for cancer cannot show its activity here..? Similar kind of practice is being reported in mythological story, in which, lord Shiva is known for this.

Keeping all these consequences aside, there must be a specific pattern to diagnose cancers which are based on several biochemical techniques. But, without knowing anything about science, medicine an illiterate person can cure cancer..! YES, this much more interesting than anything else. Now a days, many of the scientists and doctors working out to find a better way to cure cancer. Meanwhile, village person popularly called NAATI VAIDYA uses a combination of herbal leaves to cure it. There are thousands of instances where people found this medicine as more effective than the medicine prescribed by highly educated and professional doctors..!!! This again creates a head scratching doubt and remains unanswered. Many of such uneducated doctors are working to find new combination of medicines to find a better cure for cancer. Those UNNAMED DOCTORS know about the mechanism and functioning of cancer cell better than all other named doctors.  So according to me it would be a better research plan to merge modern techniques with traditional techniques. The traditional medicines should be analyzed by using modern research equipment, so that the functioning of those medicines would help us to find the target site and furthermore it would help us to identify the pathway regarding that. Add on to this discussion, it is very much essential to learn more about phytochemicals to deal with it. Phytochemicals would create a landmark in cancer research and it would be a trend setting to analyze several unknown biochemical pathways in the biological system. Ignorance towards the nature would be the biggest mistake that the scientific community is stepping and is a finest deviation from normal research tract.

People started to use pizza and burger more than the traditional food and now the younger generation is facing the severe problems including obesity, diabetes at the earliest age and even cancer. Maida flour used for the preparation of those food items are known to be very much hazardous and are slow poisons in the other way around.  Traditional Indian cuisines are purely based on naturally available food resources and are completely devoid of chemicals and adulterants. Taking all these aspects into consideration, it’s very clear that lifestyle also decides several health issues (including cancer). A lot of work has to be done which should consider lifestyle, food habit and also certain environmental condition.

Cancer forum could possibly initiate this kind of research project to motivate young minds to research.

This article is mainly based on some of the case studies and is not based on any of scientific research. This could be a true analysis with respect to some and may not be with others.

BACTERIAL TRANSPOSONS: Discovery, meaning, occurance, transposase, insertion sequence, mechanism of replicative and non replicative trasnposons.

In transposition, a transposable element, or transposon, moves from one DNA address to another. Barbara  McClintock discovered transposons in the 1940s in her studies on the genetics of maize. Since then, transposons have been found in all kinds of organisms, from bacteria to humans. We will begin with a discussion of the bacterial transposons.

Discovery of Bacterial Transposons 

James Shapiro and others laid the groundwork for the discovery of bacterial transposons with their discovery in the late 1960s of phage mutations that did not behave normally. For example, they did not revert readily the way point mutations do, and the mutant genes contained long stretches of extra DNA. Shapiro demonstrated this by taking advantage of the fact that a l phage will sometimes pick up a piece of host DNA during lytic infection of E. coli cells, incorporating the “passenger” DNA into its own genome. He allowed l phages to pick up either a wild-type E. coli galactose utilization gene (gal1) or its mutant counterpart (gal2), then measured the sizes of the recombinant DNAs, which contained l DNA plus host DNA. He measured the DNA sizes by measuring the densities of the two types of phage using cesium chloride gradient centrifugation (Chapter 20). Because the phage coat is made of protein and always has the same volume, and because DNA is much denser than protein, the more DNA the phage contains the denser it will be. It turned out that the phages harboring the gal2 gene were denser than the phages with the wild-type gene and therefore held more DNA. The simplest explanation is that foreign DNA had inserted into the gal gene and thereby inactivated it. Indeed, later experiments revealed 800–1400-bp inserts in the mutant gal gene, which were not found in the wild-type gene. In the rare cases when such mutants did revert, they lost the extra DNA. These extra DNAs that could inactivate a gene by inserting into it were the first transposons discovered in bacteria. They are called insertion sequences (ISs).

Insertion Sequences: 

The Simplest Bacterial Transposons 

Bacterial insertion sequences contain only the elements necessary for transposition. The first of these elements is a set of special sequences at a transposon’s ends, one of which is the inverted repeat of the other. The second element is the set of genes that code for the enzymes that catalyze transposition. Because the ends of an insertion sequence are inverted repeats, if one end of an insertion sequence is 59-ACCGTAG, the other end of that strand will be the reverse complement: CTACGGT-39. The inverted repeats given here are hypothetical and are presented to illustrate the point. Typical insertion sequences have somewhat longer inverted repeats, from 15 to 25 bp long. IS1, for example, has inverted repeats 23 bp long. Larger transposons can have inverted repeats hundreds of base pairs long. Stanley Cohen provided one graphic demonstration of inverted repeats at the ends of a transposon with the experiment illustrated in figure. He started with a plasmid containing a transposon with the structure shown on the figure. The original plasmid was linked to the ends of the transposon, which were inverted repeats. Cohen reasoned that if the transposon really had inverted repeats at its ends, he could separate the two strands of the recombinant plasmid, and get the inverted repeats on one strand to base-pair with each other, forming a stem-loop structure as shown on the right in figure. The stems would be double-stranded DNA composed of the two  inverted repeats: the loops would be the rest of the DNA in single-stranded form. The electron micrograph in figure shows the expected stem-loop structure. The main body of an insertion sequence codes for at least two proteins that catalyze transposition. These proteins are collectively known as transposase; we will discuss their mechanism of action later in this chapter. We know that these proteins are necessary for transposition because mutations in the body of an insertion sequence can render that transposon immobile. One other feature of an insertion sequence, shared with more complex transposons, is found just outside the transposon itself. This is a pair of short direct repeats in the DNA immediately surrounding the transposon. These repeats did not exist before the transposon inserted; they result from the insertion process itself and tell us that the transposase cuts the target DNA in a staggered fashion rather than with two cuts right across from each other. Figure shows how staggered cuts in the two strands of the target DNA at the site of insertion lead automatically to direct repeats. The length of these direct repeats depends on the distance between the two cuts in the target DNA strands. This distance depends in turn on the nature of the insertion sequence. The transposase of IS1 makes cuts 9 bp apart and therefore generates direct repeats that are 9 bp long.

More Complex Transposons 

Insertion sequences and other transposons are sometimes called “selfish DNA,” implying that they replicate at the expense of their hosts and apparently provide nothing useful in return. However, some transposons do carry genes that are valuable to their hosts, the most familiar being genes for antibiotic resistance. Not only is this a clear benefit to the bacterial host, it is also valuable to molecular biologists, because it makes the transposon much easier to track. For example, consider the situation in figure, in which we start with a donor plasmid containing a gene for kanamycin resistance (Kanr) and harboring a transposon (Tn3) with a gene for ampicillin resistance (Ampr); in addition, we have a target plasmid with a gene for tetracycline resistance (Tetr). After transposition, Tn3 has replicated and a copy has moved to the target plasmid. Now the  target plasmid confers both tetracycline and ampicillin  resistance, properties that we can easily monitor by transforming antibiotic-sensitive bacteria with the target plasmid and growing these host bacteria in medium containing both antibiotics. If the bacteria survive, they must have taken up both antibiotic resistance genes; therefore, Tn3 must have transposed to the target plasmid.

Mechanisms of Transposition 

Because of their ability to move from one place to another, transposons are sometimes called “jumping genes.” However, the term is a little misleading because it implies that the DNA always leaves one place and jumps to the other. This mode of transposition does occur and is called non replicative transposition (or “cut and paste”) because both strands of the original DNA move together from one place to the other without replicating. However, transposition frequently involves DNA replication, so one copy of the transposon remains at its original site as another copy inserts at the new site. This is called replicative transposition (or “copy and paste”) because a transposon moving by this route also replicates itself. Let us discuss how both kinds of transposition take place.

Replicative Transposition of Tn3  

Tn3, whose structure is shown in figure, illustrates one well-studied mechanism of transposition. In addition to the bla gene, which encodes ampicillin-inactivating b-lactamase, Tn3 contains two genes that are instrumental in transposition. Tn3 transposes by a two-step process, each step of which requires one of the Tn3 gene products. Figure shows a simplified version of the sequence of events. We begin with two plasmids; the donor, which harbors Tn3, and the target. In the first step, the two plasmids fuse, with Tn3 replication, to form a cointegrate in which they are  coupled through a pair of Tn3 copies. This step requires recombination between the two plasmids, which is catalyzed by the product of the Tn3 transposase gene tnpA. Figure 23.6 shows a detailed picture of how all four DNA strands involved in transposition might interact to form the cointegrate. Figures illustrate transposition between two plasmids, but the donor and target DNAs can be other kinds of DNA, including phage DNAs or the bacterial chromosome itself. The second step in Tn3 transposition is a resolution of the cointegrate, in which the cointegrate breaks down into two independent plasmids, each bearing one copy of Tn3. This step, catalyzed by the product of the resolvase gene tnpR, is a recombination between homologous sites on Tn3 itself, called res sites. Several lines of evidence show that Tn3 transposition is a two-step process. First, mutants in the tnpR gene cannot resolve cointegrates, so they cause formation of cointegrates as the final product of transposition. This demonstrates that the cointegrate is normally an intermediate in the reaction. Second, even if the tnpR gene is defective, cointegrates can be resolved if a functional tnpR gene is provided by another DNA molecule the host chromosome or another plasmid.

Nonreplicative Transposition  

Figures illustrate the replicative transposition mechanism, but transposition does not always work this way. Some transposons (e.g., Tn10) move without replicating, leaving the donor DNA and appearing in the target DNA. How does this occur? It may be that non replicative transposition starts out in the same way as replicative transposition, by nicking and joining strands of the donor and target DNAs, but then something different happens. Instead of replication occurring through the transposon, new nicks appear in the donor DNA on either side of the transposon. This releases the gapped donor DNA but leaves the transposon still bound to the target DNA. The remaining nicks in the target DNA can be sealed, yielding a recombinant DNA with the transposon integrated into the target DNA. The donor DNA has a double-stranded gap, so it may be lost or, as shown in figure, here, the gap may be repaired.

Transposons contain inverted terminal repeats

Generation of direct repeats in host DNA flanking a transposon

(a) The arrows indicate where the two strands of host DNA will be cut in a staggered fashion, 9 bp apart. (b) After cutting. (c) The transposon (yellow) has been ligated to one strand of host DNA at each end, leaving two 9-bp base gaps. (d) After the gaps are filled in, 9-bp repeats of host DNA (pink boxes) are apparent at each end of the transposon

 Detailed scheme of Tn3 transposition.

 Step 1: The two plasmids are nicked to form the free ends labeled a–h. Step 2: Ends a and f are joined, as are g and d. This leaves b, c, e, and h free. Step 3: Two of these remaining free ends (b and c) serve as primers for DNA replication, which is shown in a blowup of the replicating region. Step 4: Replication continues until end b reaches e and end c reaches h. These ends are ligated to complete the cointegrate. Notice that the whole transposon (blue) has been replicated. The paired res sites (purple) are shown for the first time here, even though one res site existed in the previous steps. The cointegrate is drawn with a loop in it, so its derivation from the previous drawing is clearer; however, if the loop were opened up, the cointegrate would look just like the one in Figure 23.5 (shown here at right). Steps 5 and 6 (resolution): A crossover occurs between the two res sites in the two copies of the transposon, leaving two independent plasmids, each bearing a copy of the transposon

 Non replicative transposition. 

The first two steps are just like those in replicative transposition, and the structure at the top is the same as that between steps 2 and 3. Next, however, new nicks occur at the positions indicated by the arrows. This liberates the donor plasmid minus the transposon, which remains attached to the target DNA. Filling gaps and sealing nicks completes the target plasmid with its new transposon. The free ends of the donor plasmid may or may not join. In any event, this plasmid has lost its transposon.

DNA REPAIR MECHANISMS: Nucleotide excision repair, Base excisoion repair, Mismatch repair

DNA repair system plays a significant role in maintaining the integrity of a species. Repair system are very unique and are found in almost all living systems (including bacterium to higher mammalian cell). Different methods are employed to repair the abnormal DNA. Due to the efficient functioning of the repair system the rate of mutation is very less.
On the basic of mechanism of repairing of DNA molecule it is classified into THREE major types, namely --

1. Nucleotide excision repair system
2. Base excision repair system
3. Mismatch repair system.

NUCLEOTIDE EXCISION REPAIR SYSTEM (NER):

Nucleotide excision repair (NER) operates by a cut-andpatch mechanism that removes a variety of bulky lesions, including pyrimidine dimers and nucleotides to which various chemical groups have become attached. Two distinct NER pathways can be distinguished: 

1. A transcription-coupled pathway in which the template strands of genes that are being actively transcribed are preferentially repaired. Repair of a template strand is thought to occur as the DNA is being transcribed,and the presence of the lesion may be signaled by a stalled RNA polymerase.This preferential repair pathway ensures that those genes of greatest importance to the cell, which are the genes the cell is actively transcribing, receive the highest priority on the “repair list.” 

2. A slower, less efficient global genomic pathway that corrects DNA strands in the remainder of the genome. Although recognition of the lesion is probably accomplished by different proteins in the two NER pathways (step 1), the steps that occur during repair of the lesion are thought to be very similar, as indicated in steps 2–6 of Figure. One of the key components of the NER repair machinery is TFIIH, a huge protein that also participates in the initiation of transcription.The discovery of the involvement of TFIIH established a crucial link between transcription and DNA repair, two processes that were previously assumed to be independent of one another (discussed in the Experimental Pathways,which can be accessed on the Web at www.wiley.com/college/karp). Included among the various subunits of TFIIH are two subunits (XPB and XPD) that possess helicase activity; these enzymes separate the two strands of the duplex (step 2) in preparation for removal of the lesion.The damaged strand is then cut on both sides of the lesion by a pair of endonucleases (step 3), and the segment of DNA between the incisions is released (step 4). Once excised, the gap is filled by a DNA polymerase (step 5),and the strand is sealed by DNA ligase (step 6).

Nucleotide excision repair

The following steps are depicted in the drawing and discussed in the text: 1) damage recognition in the global pathway is mediated by an XPC-containing protein complex, whereas damage recognition in the transcription-coupled pathway is thought to be mediated by a stalled RNA polymerase in conjunction with a CSB protein; 2) DNA strand separation (by XPB and XPD proteins,two helicase subunits of TFIIH); 3) incision (by XPG on the 3 side and the XPF–ERCC1 complex on the 5 side); 4) excision, (5)DNA repair synthesis (by DNA polymerase and/or );and (6) ligation (DNA ligase I).

BASE EXCISION REPAIR SYSTEM (BER):

A separate excision repair system operates to remove altered nucleotides generated by reactive chemicals present in the diet or produced by metabolism. The steps in this repair pathway in eukaryotes, which is called base excision repair(BER), are shown in Figure. BER is initiated by a DNA glycosylase that recognizes the alteration (step 1) and removes the base by cleavage of the glycosidic bond holding the base to the deoxyribose sugar (step 2). A number of different DNA glycosylases have been identified, each more-or-less specific for a particular type of altered base, including uracil (formed by the hydrolytic removal of the amino group of cytosine), 8-oxoguanine (caused by damage from oxygen free radicals), and 3-methyladenine (produced by transfer of a methyl group from a methyl donor). Structural studies of the DNA glycosylase that removes the highly mutagenic 8-oxoguanine (oxoG) indicate that this enzyme diffuses rapidly along the DNA “inspecting” each of the G-C base pairs within the DNA duplex (step 1). In step 2, the enzyme has come across an oxoG-C base pair. When this occurs, the enzyme inserts a specific amino acid side chain into the DNA helix, causing the nucleotide to rotate (“flip”) 180 degrees out of the DNA helix and into the body of the enzyme (step 2). If the nucleotide does, in fact, contain an oxoG, the base fits into the active site of the enzyme (step 3) and is cleaved from its associated sugar. In contrast, if the extruded nucleotide contains a normal guanine,which only differs in structure by two atoms from oxoG, it is unable to fit into the enzyme’s active site (step 4) and it is returned to its appropriate position within the stack of bases. Once an altered purine or pyrimidine is removed by a glycosylase, the “beheaded” deoxyribose phosphate remaining in the site is excised by the combined action of a specialized (AP) endonuclease and a DNA polymerase. AP endonuclease cleaves the DNA backbone (step 3) and a phosphodiesterase activity of polymerase  removes the sugar–phosphate remnant that had been attached to the excised base (step 4). Polymerase  then fills the gap by inserting a nucleotide complementary to the undamaged strand (step 5), and the strand is sealed by DNA ligase III (step 6). The fact that cytosine can be converted to uracil may explain why natural selection favored the use of thymine, rather than uracil, as a base in DNA, even though uracil was presumably present in RNA when it served as genetic material during the early evolution of life. If uracil had been retained as a DNA base,it would have caused difficulty for repair systems to distinguish between a uracil that “belonged”at a particular site and one that resulted from an alteration of cytosine.
 

Base excision repair

 

MISMATCH REPAIR SYSTEM (MMR):

It was noted earlier that cells can remove mismatched bases that are incorporated by the DNA polymerase and escape the enzyme’s proofreading exonuclease. This process is called mismatch repair (MMR). A mismatched base pair causes a distortion in the geometry of the double helix that can be recognized by a repair enzyme.But how does the enzyme “recognize” which member of the mismatched pair is the incorrect nucleotide? If it were to remove one of the nucleotides at random, it would make the wrong choice 50 percent of the time,creating a permanent mutation at that site.Thus,for a mismatch to be repaired after the DNA polymerase has moved past a site,it is important that the repair system distinguish the newly synthesized strand, which contains the incorrect nucleotide, from the parental strand,which contains the correct nucleotide.In E.coli, the two strands are distinguished by the presence of methylated adenosine residues on the parental strand. DNA methylation does not appear to be utilized by the MMR system in eukaryotes,and the mechanism of identification of the newly synthesized strand remains unclear.Several different MMR pathways have been identified and will not be discussed.

DISCOVERY OF tRNA: Zamecnik experiment with radio leucine & microsomal proteins.

In 1958, Francis Crick postulated the existence of an adaptor molecule, presumably RNA, that could serve as a mediator between the string of nucleotides in DNA (actually in mRNA) and the string of amino acids in the corresponding protein. Crick favored the idea that the adapter contained two or three nucleotides that could pair with nucleotides in codons, although no one knew the nature of codons, or even of the existence of mRNA, at that time. Transfer RNA had already been discovered by Paul Zamecnik and coworkers a year earlier, although they did not realize that it played an adapter role.

The Discovery of tRNA 

By 1957, Zamecnik and colleagues had worked out a cell free protein synthesis system from the rat. One of the components of the system was a so-called pH 5 enzyme fraction that contained the soluble factors that worked with ribosomes to direct translation of added mRNAs. Most of the components in the pH 5 enzyme fraction were proteins, but Zamecnik’s group discovered that this mixture also included a small RNA. Of even more interest was their finding that this RNA could be coupled to amino acids. To demonstrate this, they mixed the RNA with the pH 5 enzymes, ATP, and [14C] leucine. Figure 19.26a shows that the more labeled leucine these workers added to the mixture, the more was attached to the RNA, which they separated from protein by phenol extraction. Furthermore, when they left out ATP, no reaction occurred. We now know that this reaction was the charging of tRNA with an amino acid. Not only did Zamecnik and his coworkers show that the small RNA could be charged with an amino acid, they also demonstrated that it could pass its amino acid to a growing protein. They performed this experiment by mixing the [14C]leucine-charged pH 5 RNA with microsomes— small sections of endoplasmic reticulum containing ribosomes. Figure 19.26b shows a near-perfect correspondence between the loss of radioactive leucine from the pH 5 RNA and gain of the leucine by the protein in the microsomes. This represented the incorporation of leucine from leucyltRNA into nascent polypeptides on ribosomes.

Discovery of tRNA. 

tRNAs can be charged with leucine. Zamecnik and colleagues added labeled leucine to the tRNAcontaining fraction and plotted the binding of leucine to the RNA as a function of labeled leucine added.

 Discovery of tRNA 

The charged tRNA can donate its amino acid to nascent protein. Zamecnik and colleagues followed the radioactivity (cpm) lost from the RNA (blue) and gained by the nascent proteins (red) in the microsomes, which contained the ribosomes. The reciprocal relationship between these curves suggested that the RNA was donating its amino acid to the growing protein.