Coordination of DNA synthesis and replicative unwinding by the S-phase checkpoint pathways

Nedelcheva-Veleva, Marina N.; Krastev, Dragomir B.; Stoynov, Stoyno

doi:10.1093/nar/gkl528

Cited by 16 publications

(15 citation statements)

References 101 publications

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“…Replisome deregulation can lead to genome instability, both through replication fork collapse and the generation of single strand gaps created by the uncoupling of polymerases from helicases (1,6,55). In this study, we find that Tim, a regulator of DNA replication efficiency (24,25,27,28), suppresses DSBs and chromosome abnormalities during unperturbed DNA synthesis.…”

Section: Discussionmentioning

confidence: 57%

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication

Urtishak

Smith

Chanoux

et al. 2009

Journal of Biological Chemistry

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Genome integrity is maintained during DNA replication by coordination of various replisome-regulated processes. Although it is known that Timeless (Tim) is a replisome component that participates in replication checkpoint responses to genotoxic stress, its importance for genome maintenance during normal DNA synthesis has not been reported. Here we demonstrate that Tim reduction leads to genomic instability during unperturbed DNA replication, culminating in increased chromatid breaks and translocations (triradials, quadriradials, and fusions). Tim deficiency led to increased H2AX phosphorylation and Rad51 and Rad52 foci formation selectively during DNA synthesis and caused a 3-4-fold increase in sister chromatid exchange. The sister chromatid exchange events stimulated by Tim reduction were largely mediated via a Brca2/Rad51-dependent mechanism and were additively increased by deletion of the Blm helicase. Therefore, Tim deficiency leads to an increased reliance on homologous recombination for proper continuation of DNA synthesis. Together, these results indicate a pivotal role for Tim in maintaining genome stability throughout normal DNA replication.DNA synthesis utilizes complex sets of genes and functionalities to prevent genome maintenance failures. These potential failures include the misincorporation of nucleotides, the collapse of replication forks, and the formation of secondary structures that lead to chromosome deletions, duplications, and other mutagenic events (1-4).In addition to polymerases and their cofactors, several higher order protein complexes act in concert during DNA replication to promote chromatin decondensation, DNA duplex unwinding, and protection of the resulting single-stranded DNA (ssDNA).2 Failure to efficiently coordinate these processes can lead to stalling and collapse of the replication fork into double strand breaks (DSBs). For example, in budding yeast, deficiencies in DNA priming caused by low levels of pol ␣ lead to a 22-fold increase in mitotic recombination (5). Similarly, in vertebrate cells, treatment with DNA polymerase inhibitors (e.g. aphidicolin) causes the generation of short segments of ssDNA, via polymerase-helicase uncoupling (6 -11), and increases chromatid breaks at common fragile sites (12). The importance of polymerase processivity in genome stabilization raises the question to what degree the other components of the replication apparatus participate in genome maintenance. Tim and its putative orthologs in yeast (Swi1 in Schizosaccharomyces pombe and Tof1 in Saccharomyces cerevisiae) facilitate DNA replication. Although less is known about Tim function in mammals, several functions of Swi1 and Tof1 have recently been described. Swi1 and Tof1 associate with chromatin and travel with the replication fork during S phase and are required for normal pausing at replication fork barriers (13)(14)(15)(16)(17)(18)(19)(20). In addition, Tof1 associates with Cdc45 and MCM6, and deletion of Tof1 can lead to slowed replication fork progression (13,17,21).Importantly, recen...

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Section: Discussionmentioning

confidence: 57%

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication

Urtishak

Smith

Chanoux

et al. 2009

Journal of Biological Chemistry

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“…We find that a SET2 deletion suppresses the HU sensitivity of cdc2-1 and ctf4 mutations and a CHD1 deletion suppresses the HU sensitivity in orc2-1 and mec1 sml1 strains. CDC2 encodes the catalytic subunit of DNA polymerase-d, CTF4 is required for efficient sister chromatid cohesion (Hanna et al 2001) and competes with yFACT for binding to DNA polymerase-a (Wittmeyer and Formosa 1997), ORC2 encodes a subunit of the origin recognition complex (DaSilva and Duncker 2007), and MEC1 encodes the checkpoint kinase that monitors replication fork integrity (Nedelcheva-Veleva et al 2006). Suppression of these replication-defective mutations by set2 and chd1 strongly implies that these chromatin-modifying factors have specific roles in negatively regulating DNA replication.…”

Section: Discussionmentioning

confidence: 99%

A Role for Chd1 and Set2 in Negatively Regulating DNA Replication in Saccharomyces cerevisiae

Biswas¹,

Takahata²,

Hua

et al. 2008

Genetics

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Chromatin-modifying factors regulate both transcription and DNA replication. The yFACT chromatinreorganizing complex is involved in both processes, and the sensitivity of some yFACT mutants to the replication inhibitor hydroxyurea (HU) is one indication of a replication role. This HU sensitivity can be suppressed by disruptions of the SET2 or CHD1 genes, encoding a histone H3(K36) methyltransferase and a chromatin remodeling factor, respectively. The additive effect of set2 and chd1 mutations in suppressing the HU sensitivity of yFACT mutants suggests that these two factors function in separate pathways. The HU suppression is not an indirect effect of altered regulation of ribonucleotide reductase induced by HU. set2 and chd1 mutations also suppress the HU sensitivity of mutations in other genes involved in DNA replication, including CDC2, CTF4, ORC2, and MEC1. Additionally, a chd1 mutation can suppress the lethality normally caused by disruption of either MEC1 or RAD53 DNA damage checkpoint genes, as well as the lethality seen when a mec1 sml1 mutant is exposed to low levels of HU. The pob3 defect in S-phase progression is suppressed by set2 or chd1 mutations, suggesting that Set2 and Chd1 have specific roles in negatively regulating DNA replication.

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“…A3s must compete with RPA to access to the ssDNA substrate [199,200]. Thus, in a normal cell, A3 must access ssDNA between the polymerase and helicase, which are usually traveling along the DNA rapidly [200,201] (Figure 1.10). Although there are ssDNA gaps on the lagging strand, which is probably why this strand is favored by A3 enzymes, access to the ssDNA on the lagging strand also requires the A3 to displace RPA (Figure 1.10).…”

Section: Role Of Apobec In Somatic Mutagenesismentioning

confidence: 99%

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

2018

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Apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3 (APOBEC3) enzymes are a family of single-stranded (ss)DNA cytosine deaminases that serve as a host restriction factors for retrotransposons and viruses that contain a ssDNA intermediate. While the APOBEC3 family was originally expanded to restrict endogenous retroelements, the majority of these targets are now inactivated and the family has been found to act on different targets, such as viral ssDNA as a mechanism of viral defense. Some of the family members also catalyze "offtarget" deaminations in human ssDNA and have been implicated in somatic mutagenesis that can lead to cancer.My PhD thesis research investigated the biochemical mechanisms underlying both the benefits and the risks of the A3 family of enzymes. The central hypothesis of this work is that A3 enzymes have evolved distinct biochemical mechanisms to deaminate ssDNA that differentiate A3 restriction of retroelements and retroviruses from A3-induced somatic mutagenesis.Therefore, we investigated the biochemical mechanisms the A3 enzymes utilize during restriction of Human Immunodeficiency Virus (HIV) in both deamination-dependent and deaminationindependent modes, as well as the unique mechanisms employed during mutation of genomic DNA. There are seven APOBEC3 members (A-H, excluding E) and of these, four members (A3D, A3F, A3G, A3H) have been identified to restrict HIV replication in CD4 T+ cells, and currently three members (A3A, A3B and A3H haplotype I) have been implicated in somatic mutagenesis.The APOBEC3 enzymes that restrict HIV replication function optimally in the absence of the HIV viral infectivity factor (Vif). Vif targets APOBEC3 for degradation via the proteasome in HIV infected cells. For APOBEC3s that are able to fortuitously escape Vif mediated degradation and become encapsidated, the enzymes can deaminate cytosines to form uracils in viral (-)DNA. Replication of (-)DNA to (+)DNA causes HIV-1 reverse transcriptase to incorporate adenines opposite uracils which creates C/G→T/A transition mutations. While restriction of HIV most commonly occurs through this deamination-dependent mechanism, APOBEC3s have also been identified to interfere with HIV reverse transcriptase processes in a deamination-independent manner, although this mechanism is secondary to the deaminationdependent mode. In order to restrict retroelements and viruses such as HIV, the A3 enzymes iii require an efficient processive ssDNA scanning mechanism that allows them to search for, and locate cytosines on ssDNA in the finite amount of time the substrate is available during formation of the double-stranded DNA provirus. To understand if this requirement was lost in A3 enzymes unable to restrict HIV, we studied A3C. Human A3C is not able to restrict HIV, in comparison to the more active gorilla and chimpanzee A3C orthologs that can restrict HIV, however an explanation for this difference was lacking. A polymorphism, S188I, in human A3C, which is found in less than 3% of the global population lead...

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Coordination of DNA synthesis and replicative unwinding by the S-phase checkpoint pathways

Cited by 16 publications

References 101 publications

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication

A Role for Chd1 and Set2 in Negatively Regulating DNA Replication in Saccharomyces cerevisiae

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

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