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Chapter 11 : Alternative Excision Repair of DNA
Category: Microbial Genetics and Molecular Biology
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This chapter discusses repair pathways of DNA such as alternative excision repair (AER). An AER pathway that has been most extensively defined by studies of E. coli involves a specific endonuclease called endonuclease V. Endonuclease V-mediated incision of DNA is presumably followed by other biochemical events that complete the excision repair of deaminated bases and other substrates. Early studies demonstrated enzymatic activity primarily on DNA containing deoxyinosine, a product of the deamination of deoxyadenosine. This activity was thus designated deoxyinosine 3’ endonuclease of E. coli. A series of genetic studies indicate that endonuclease V of E. coli also incises DNA containing the purine analog N-6-hydroxylaminopurine (HAP). Then, the chapter talks about AER mediated by other endonucleases. It is evident from the examples provided in the chapter that nature has been versatile in evolving mechanisms that afford the repair of various types of spontaneous and environmentally generated base damage in DNA. In summary, the chapter provides a cogent reminder that DNA damage is a pervasive phenomenon in living cells and it is obvious that there is no dearth of strategies adopted by nature to mitigate the lethal and mutagenic effects of such damage.
Endonuclease V of E. coli attacks uracil-containing DNA, generating acid-soluble products. Solid black line, DNA from phage PBS2 in which thymine is fully replaced by U; broken black line, phage T5 DNA with 12.5% replacement of T by U; solid gold line, phage T5 DNA with 3.8% replacement of T by U; broken gold line, phage T5 DNA with <0.3% replacement of T by U. (Adapted from reference 9 .)
Endonuclease V of E. coli attacks DNA containing deoxyinosine (dI) or deoxyxanthosine (dX). Different amounts of endonuclease V were incubated with double-stranded (ds) dI:dC (lanes 1 to 5), single-stranded dI (ss) (lanes 6 to 10), double-stranded dX:dC (lanes 11 to 15), and single-stranded dX (lanes 16 to 20). In each case the oligonucleotides were 5’-end radiolabeled and degradation products were identified by autoradiography following gel electrophoresis. (Adapted from reference 16 .)
Endonuclease V (Nfi) cuts the sugar-phosphate backbone of a DNA strand at the second nucleotide 3’ to the substrate base, leaving 3’ OH and 5’ phosphate termini.
(A) Alignment of amino acid sequences of human, mouse, and E. coli (Nfi) endonuclease V, showing identical and similar amino acids. (B) Conserved amino acids in human, mouse, and E. coli (Nfi) endonuclease V and those in E. coli UvrC protein (UvrC). (Adapted from reference 31 .)
An nfi mutant strain of E. coli defective for endonuclease V is not abnormally sensitive to killing following exposure to various DNA-damaging agents (hydrogen peroxide [A], γ-rays [B], UV radiation [C], and nitrous acid [D] are shown), even in the presence of other mutations that confer sensitivity to these agents. Other mutant strains shown are xth (exonuclease III) and nfo (endonuclease IV). wt, wild type. (Adapted from reference 15 .)
A•T → G•C transition mutations are induced in an nfi mutant strain of E. coli following exposure to sodium nitrate (NaNO3) or sodium nitrite (NaNO2). (Adapted from reference 40 .)
Suggested mechanism of the alternate excision repair of inosine (I) in DNA in mammalian cells. Excision repair is initiated by endonucleolytic cleavage by endonuclease V (ENDO V) at the second phosphodiester bond 3’ to the inosine moiety. The nucleotide may be removed by a suitable exonuclease, such as the 3’ mismatch-specific exonuclease activity of the AP endonuclease APEX 1 (see chapter 6) or the 3’ flap endonuclease activity of MUS81-MMS4 protein (see chapter 19). (Adapted from reference 31 .)
The Uve1 endonuclease from S. pombe incises duplex oligonucleotides containing either a CPD or a (6–4) PP directly 5’ to the lesions. (A) 5’-End-labeled duplex oligonucleotides containing either a (6–4)PP or a CPD were incubated with Uve1 (lanes 3 and 7) or phage T4 PD-DNA glycosylase/AP lyase (T4 denV) (lanes 2 and 6), subjected to hot-alkali treatment (lanes 4 and 5), or left untreated (lanes 1 and 8). The scission products were resolved on a denaturing polyacrylamide DNA sequencing gel. Sequencing reaction products (GA and CT) are shown in the two leftmost lanes. The nucleotide sequence of the oligonucleotide, numbered from the 5’ terminus, is shown. The bracketed TT residues at positions 21 and 22 represent either a CPD or a (6–4)PP. Note that Uve1 endonuclease cuts DNA containing either photoproduct, while the T4 enzyme is specific for CPD. (B) Endonucleolytic cleavage by the SPDE enzyme is dependent on the presence of a photoproduct in the oligonucleotide. When the CPD is repaired by prior treatment with pyrimidine dimer-DNA photolyase (lanes 2 and 4), no cleavage is observed. (C) Comparison of the mode of incision of DNA at a CPD site by SPDE (Uve1) and T4 denV. (Adapted from reference 5 .)
The uve1+ gene (black line) can rescue the UV radiation sensitivity of an E. coli phr recA uvrA triple mutant (gold line) defective in photoreactivation, recombination, and nucleotide excision repair. (Adapted from reference 38 .)
Uve1 partially rescues the sensitivity of an E. coli strain (xth nfo) defective in AP endonuclease activity (compare the dark and light gold lines indicating the presence or absence of the uve1+ gene, respectively) following exposure of cells to MMS (A) or t-butyl hydroperoxide (tBuO2H) (B). The black line indicates the survival of a wild-type E. coli strain. (Adapted from reference 21 .)
Schematic representation of suggested alternative pathways for the excision repair of base damage involving the Uve1 endonuclease of S. pombe. The Rad2 subpathway involves a flap endonuclease (see chapter 6), and the recombination subpathway involves proteins required for recombination and recombinational repair (see chapter 19). (Adapted from reference 30 .)