Translesion synthesis (TLS) employs specialized polymerases to incorporate nucleotides opposite DNA lesions, allowing replication to continue temporarily without repairing the lesions, while sacrificing replication fidelity (1). TLS depends on a ubiquitin molecule attached to the clamp PCNA at residue Lys164 (2, 3); the precise biochemical and molecular basis how PCNA modification stimulates TLS is still elusive (4-10). In this study, we discovered that DNA polymerase δ (Pol δ) 3′ to 5′ proofreading exonuclease degrades nascent TLS synthesis opposite pyrimidine dimers and inhibits TLS on both leading and lagging strands. Rad18 E3 ligase and PCNA ubiquitylation become dispensable for TLS when Pol δ exonuclease is inactive. Biochemical reconstitution showed that the major UV lesion bypass polymerase Rad30 is intrinsically inefficient at bypassing thymidine dimers and requires multiple turnovers. Pol δ exonuclease impedes Rad30 activity both catalytically and non-catalytically during and post-lesion bypass. Importantly, PCNA and PCNA ubiquitylation enhance processivity of Rad30 after thymidine dimer bypass, a critical step for further DNA synthesis extension and overcoming Pol δ inhibition. Our study reveals a new paradigm how PCNA ubiquitylation facilitates TLS and the new role of Pol δ nuclease in lesion bypass pathway choice and replication fidelity at fork-blocking DNA lesions. ### Competing Interest Statement The authors have declared no competing interest.

In this work, we developed a DNA dosimeter, consisting of 4-kb DNA strands attached to magnetic streptavidin beads and labeled with fluorescein, to detect double-strand breaks (DSBs). The purpose here was to evaluate whether the DNA dosimeter readings reflect the relative biological effects of 160 kVp and 6 MV X rays. AVarian 600 C/D linac (6 MV) and a Faxitron cabinet X-ray system (160 kVp), both calibrated using traceable methods, were used to deliver high- and low-energy photons, respectively, to DNA dosimeters and multiple cell lines (mNs-5, HT-22 and Daoy). The responses were fit versus dose, and were used to quantify the dose of low-energy photons that produced the same response as that of the high-energy photons, at doses of 3, 6 and 9 Gy. The equivalent doses were utilized to calculate the relative biological effectiveness (RBEDSB and RBEcell survival). Additionally, a neutral comet assay was performed to measure the amount of intracellular DNA DSB, and ultimately the RBEcomet assay. The results of this work showed 160-kVp photon RBE values and 95% confidence intervals of 1.12 ± 0.04 (mNS-5), 1.16 ± 0.06 (HT-22), 1.25 ± 0.09 (Daoy) and 1.21 ± 0.24 (DNA dosimeter) at 9 Gy and 1.32 ± 0.16 (comet assay) at 3 Gy. Within the current error, the DNA dosimeter measured RBEDSB values in agreement with the RBEcell survival and assay from the cell survival and comet assay RBEcomet measurements. These results suggest that the DNA dosimeter can measure the changes in the radiobiological effects from different energy photons.

Ribonucleoside monophosphates (rNMPs) mis-incorporated during DNA replication are removed by RNase H2-dependent excision repair or by topoisomerase I (Top1)-catalyzed cleavage. The cleavage of rNMPs by Top1 produces 3' ends harboring terminal adducts, such as 2',3'-cyclic phosphate or Top1 cleavage complex (Top1cc), and leads to frequent mutagenesis and DNA damage checkpoint induction. We surveyed a range of candidate enzymes from Saccharomyces cerevisiae for potential roles in Top1-dependent genomic rNMP removal. Genetic and biochemical analyses reveal that Apn2 resolves phosphotyrosine-DNA conjugates, terminal 2',3'-cyclic phosphates, and their hydrolyzed products. APN2 also suppresses 2-base pair (bp) slippage mutagenesis in RNH201-deficient cells. Our results define additional activities of Apn2 in resolving a wide range of 3' end blocks and identify a role for Apn2 in maintaining genome integrity during rNMP repair.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.

Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.