Sunday, 26 April 2015

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.







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