Yeast can be readily transformed by single-stranded oligonucleotides (ssOligos). Previously, we showed that an ssOligo that generates a 1-nt loop containing an AP site corrected the −1 frameshift mutation in the lys2ΔA746 allele. However, these experiments had to be performed in yeast apn1 mutants lacking the major AP endonuclease. In this study, we show that bypass of an AP site can be studied in repair-proficient yeast by using ssOligos that generates a 7-nt loop containing an AP site. The bypass studies performed using the ssOligos that generate a 7-nt loop was validated by demonstrating that the result obtained are similar to those derived using ssOligos containing a 1-nt loop in an apn1 mutant. By using the 7-nt loop system, we showed that the bypass efficiencies of AP sites are dependent on the sequence context that surrounds the lesion and are apparently no affected by the direction of DNA replication. In contrast, the mutagenic specificity of an AP site is not affected by the sequence context or the direction of replication. In all cases, dC is inserted at twice the frequency of dA opposite an AP site, indicating that REV1 is mainly responsible for bypass of AP sites at all lesion sites studied.
Transformation of both prokaryotes and eukaryotes with single-stranded oligonucleotides can transfer sequence information from the oligonucleotide to the chromosome. We have studied this process using oligonucleotides that correct a −1 frameshift mutation in the LYS2 gene of Saccharomyces cerevisiae. We demonstrate that transformation by oligonucleotides occurs preferentially on the lagging strand of replication and is strongly inhibited by the mismatch-repair system. These results are consistent with a mechanism in which oligonucleotides anneal to single-stranded regions of DNA at a replication fork and serve as primers for DNA synthesis. Because the mispairs the primers create are efficiently removed by the mismatch-repair system, single-stranded oligonucleotides can be used to probe mismatch-repair function in a chromosomal context. Removal of mispairs created by annealing of the single-stranded oligonucleotides to the chromosomal DNA is as expected, with 7-nt loops being recognized solely by MutSβ and 1-nt loops being recognized by both MutSα and MutSβ. We also find evidence for Mlh1-independent repair of 7-nt, but not 1-nt, loops. Unexpectedly, we find a strand asymmetry of mismatch-repair function; transformation is blocked more efficiently by MutSα on the lagging strand of replication, whereas MutSβ does not show a significant strand bias. These results suggest an inherent strand-related difference in how the yeast MutSα and MutSβ complexes access and/or repair mismatches that arise in the context of DNA replication.
The human endonuclease V gene is located in chromosome 17q25.3 and encodes a 282 amino acid protein that shares about 30% sequence identity with bacterial endonuclease V. This study reports biochemical properties of human endonuclease V with respect to repair of deaminated base lesions. Using soluble proteins fused to thioredoxin at the N-terminus, we determined repair activities of human endonuclease V on deoxyinosine (I)-, deoxyxanthosine (X)-, deoxyoxanosine (O)- and deoxyuridine (U)-containing DNA. Human endonuclease V is most active with deoxyinosine-containing DNA but with minor activity on deoxyxanthosine-containing DNA. Endonuclease activities on deoxyuridine and deoxyoxanosine were not detected. The endonuclease activity on deoxyinosine-containing DNA follows the order of single-stranded I > G/I > T/I > A/I > C/I. The preference of the catalytic activity correlates with the binding affinity of these deoxyinosine-containing DNAs. Mg 2+ and to a much less extent, Mn 2+ , Ni 2+ , Co 2+ can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells deficient in nfi, mug and ung genes caused three-fold reduction in mutation frequency. This is the first report of deaminated base repair activity for human endonuclease V. The relationship between the endonuclease activity and deaminated deoxyadenosine (deoxyinosine) repair is discussed.
Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strand-specific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.
It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3'-terminal 8 oxoG, unlike a 3'-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.
5'-R and 5'-S diastereoisomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24-32bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a] P-N 2 -dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N 2 -dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.