A top-down view on DNA replication and recombination from 9,000 feet above sea level

A report of the Keystone Symposium 'DNA Replication and Recombination' held in Keystone, USA, 27 February to 4 March 2011

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion

Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage

The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases ∼6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency ∼3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase ε does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19–27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions

Shortage of dNTPs underlies altered replication dynamics and DNA breakage in the absence of the APC/C cofactor Cdh1

This research was funded by grants from the Spanish Ministry of Economy and Competitiveness MINECO (CSD2007-0015, BFU2011-28274 and BFU2014-55439) and Junta de Castilla y León (CSI151U13 and CSI084U16), the Swedish Cancer Society, the Knut and Alice Wallenberg Foundation and the Swedish Research Council (A.C.). I.G.H is supported by Fundación Científica de la Asociación Española contra el Cáncer (AECC). J.G. and R.R were recipients of CSIC JAE and FPU predoctoral fellowships (MINECO)Peer reviewedPostprin

Elevated dNTP levels suppress hyper-recombination in Saccharomyces cerevisiae S-phase checkpoint mutants

MEC1, the essential yeast homolog of the human ATR/ATM genes, controls the S-phase checkpoint and prevents replication fork collapse at slow zones of DNA replication. The viability of hypomorphic mec1-21 is reduced in the rad52 mutant, defective in homologous recombination, suggesting that replication generates recombinogenic lesions. We previously observed a 6-, 10- and 30-fold higher rate of spontaneous sister chromatid exchange (SCE), heteroallelic recombination and translocations, respectively, in mec1-21 mutants compared to wild-type. Here we report that the hyper-recombination phenotype correlates with lower deoxyribonucleoside triphosphate (dNTP) levels, compared to wild-type. By introducing a dun1 mutation, thus eliminating inducible expression of ribonucleotide reductase in mec1-21, rates of spontaneous SCE increased 15-fold above wild-type. All the hyper-recombination phenotypes were reduced by SML1 deletions, which increase dNTP levels. Measurements of dNTP pools indicated that, compared to wild-type, there was a significant decrease in dNTP levels in mec1-21, dun1 and mec1-21 dun1, while the dNTP levels of mec1-21 sml1, mec1-21 dun1 sml1 and sml1 mutants were ∼2-fold higher. Interestingly, higher dNTP levels in mec1-21 dun1 sml1 correlate with ∼2-fold higher rate of spontaneous mutagenesis, compared to mec1-21 dun1. We suggest that higher dNTP levels in specific checkpoint mutants suppress the formation of recombinogenic lesions

Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex

DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2–7-GINS(Psf1–3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the di�vision labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1–1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1–1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome

Pre-activation of the genome integrity checkpoint increases DNA damage tolerance

The genome integrity checkpoint is a conserved signaling pathway that is regulated in yeast by the Mec1 (homologous to human ATR) and Rad53 (homologous to human Chk1) kinases. The pathway coordinates a multifaceted response that allows cells to cope with DNA damage and DNA replication stress. The full activation of the checkpoint blocks origin firing, stabilizes replication forks, activates DNA repair proteins and may lead to senescence or apoptosisin higher eukaryotes. We have recently demonstrated that endogenous replication stress can activate the genome integrity checkpoint in budding yeast at a low level that does not go so far as to interfere with cell cycle progression, but it does activate DNA damage-inducible proteins. Here we demonstrate that the low level pre-activation of the checkpoint, either by endogenous replication stress or by the nucleotide-depleting drug hydroxyurea, can increase damage tolerance to multiple DNA-damaging agents. These results may provide new strategies for using the checkpoint to protect normal cells from genotoxic stress