Apn1 and Apn2 endonucleases prevent accumulation of repair-associated DNA breaks in budding yeast as revealed by direct chromosomal analysis

Abstract

Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).

Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

“…All yeast mutants used were derived from haploid strains containing a circular form of chromosome III (Chr III) (23). We first determined survival after UV-C irradiation (50 J/m 2 ) of cells that were nocodazole-arrested in the G2 stage of the cell cycle (>90% arrest; see Table S1).…”

“…Yeast strains used in this study are derivatives of two isogenic haploid strains, MWJ49 and MWJ50 (MATα leu2-3,112 ade5-1 his7-2 ura3Δ trp1-289), which contain a circular form of Chr III (23). The deletion mutants rad30, rev3, rad50, rad51, rad52, exo1, and their derived double or triple mutants were created by replacing the relevant ORF in these strains with selectable markers as previously described (55).…”

“…Detection of UV-induced damage and repair were based on PFGE analysis as previously described (23). Briefly, control and UV-treated cells were mixed with low-melting agarose [2% (wt/vol) stock in Tris-EDTA buffer, final concentration 0.6%] containing 1 mg/mL Zymolyase (100 U/mg) (MP Biochemicals) to prepare agarose-embedded DNA plugs.…”

“…The requirement for HR in the absence of TLS could be caused by the generation of DSBs, possibly through overlapping repair regions as previously suggested (32,33) or somehow during the generation of the ssDNAs. Using our previously described circular chromosome assay that can detect random DSBs efficiently at frequencies of only a few DSBs per yeast genome (18,23), we investigated whether TLS deficiency leads to increased DSBs during the processing of UV damage. In this assay, a circular Chr III, which is ∼300 kb, is retained in the well during PFGE.…”

“…Because DSBs are expected to accumulate in the HR mutants, these results suggest that there is little generation of DSBs. Previously, we had estimated that the background spontaneous level of DSBs in nongrowing cells is around one or two DSBs per yeast genome (23). Based on a two-to three-fold increase, at most, in band density after UV irradiation (Fig.…”