Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast

V. Pages; S. R. Santa Maria; L. Prakash; S. Prakash

doi:10.1101/gad.1793409

The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage

Cell Division ◽

10.1186/s13008-021-00072-x ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Muhseena N. Katheeja ◽

Shankar Prasad Das ◽

Suparna Laha

Keyword(s):

Dna Damage ◽

Budding Yeast ◽

S Phase ◽

Wild Type ◽

Yeast Protein ◽

Repair Gene ◽

Replication Checkpoint ◽

Bud Emergence ◽

Checkpoint Pathway

Abstract Background The budding yeast protein Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This helicase is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with faster kinetics in chl1 mutants compared to wild-type cells. Also, more damage to DNA is observed in chl1 cells. The viability falls synergistically in rad24chl1 cells. The regulation of Chl1p on budding kinetics in G1 phase falls in line with Rad9p/Chk1p and shows a synergistic effect with Rad24p/Rad53p. rad9chl1 and chk1chl1 shows similar bud emergence as the single mutants chl1, rad9 and chk1. Whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24, rad53 and chl1. In presence of MMS induced damage, synergistic with Rad24p indicates Chl1p’s role as a checkpoint at G1/S acting parallel to damage checkpoint pathway. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further, we have also confirmed that the checkpoint defect functions in parallel to the damage checkpoint pathway of Rad24p. Conclusion Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint activity in presence of damage. This confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1p thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p for Rad53p activation when damaging agents perturb the DNA. Apart from checkpoint activation, it also regulates the budding kinetics as a repair gene.

Download Full-text

The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage.

10.21203/rs.3.rs-151524/v1 ◽

2021 ◽

Author(s):

Katheeja Muhseena N. ◽

Shankar Prasad Das ◽

Suparna Laha

Keyword(s):

Dna Damage ◽

Budding Yeast ◽

S Phase ◽

Wild Type ◽

Yeast Protein ◽

Replication Checkpoint ◽

Bud Emergence ◽

Checkpoint Pathway ◽

M Phase

Abstract Background: The helicase Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This budding yeast protein is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results: G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with a faster kinetics in chl1 mutants compared to wild-type cells. This role of Chl1p in G1 phase is Rad9p dependent and independent of Rad24 and Rad53. rad9chl1 shows similar bud emergence as the single mutants chl1 and rad9 whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24 , rad53 and chl1 . In case of damage induced by genotoxic agent like hydroxyurea, Chl1p acts as a checkpoint at G1/S. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further we have observed that the checkpoint defect is synergistic with the replication checkpoint Sgs1p and functions in prallel to the checkpoint pathway of Rad24p. Conclusion: Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint, confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1 thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p as well as Rad53p activation when damaging agents perturbs the DNA.

Download Full-text

Regulation of PCNA Mono-Ubiqutination by FANCD2 and Rad51 in Response to Hydroxyurea.

Blood ◽

10.1182/blood.v120.21.2377.2377 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2377-2377

Author(s):

Gary M. Kupfer ◽

Xiaoyong Chen

Keyword(s):

Dna Damage ◽

Ubiquitin Ligase ◽

Conflicts Of Interest ◽

Translesion Synthesis ◽

Deficient Mutant ◽

Independent Manner ◽

Knock Down ◽

Mutant Cells

Abstract Abstract 2377 FANCD2 is a key player in FA pathway. It has been shown that FANCD2 can interact with PCNA and with Rad18, the ubiquitin ligase responsible for PCNA mono-ubiquitination. The mono-ubiquitination of PCNA is very important for its function in translesion synthesis. We found that in response to DNA damage agent hyxdroxyurea (HU) the interaction of FANCD2 with Rad18 or Rad51, and the interaction of Rad51 with Rad18 or PCNA was enhanced. FANCD2 is required for increased interaction between Rad51 and Rad18 indicating that FANCD2, Rad51 and Rad18 form a complex in response to HU. Rad18 was required for PCNA mono-ubiquitination in response to HU. FANCD2 deficient cells failed to enhance the interaction between Rad18 and Rad51. Furthermore, PCNA mono-ubiquitination was impaired in FANCD2 deficient cells in response to HU. FANCD2 mono-ubiquitination deficient mutant partially rescued PCNA mono-ubiquitination. The partial mono-ubiquitination of PCNA in response to HU in FANCA deficient mutant confirmed the role of non-ubiquitinated FANCD2 in PCNA mono-ubiquitination. The normal mono-ubiquitination of PCNA in FANCJ deficient mutant confirmed that the effect of FANCD2 in PCNA mon-ubiquitination is not due to FA pathway deficiency. Rad51 was also involved in regulating PCNA mono-ubiquitination in response to HU. Rad51 siRNA knock down cells showed decreased PCNA mono-ubiquitination in response to HU. The role of Rad51 in regulating PCNA mono-ubiquitination did not require BRCA1, indicating that this function is independent of HR. More importantly FANCD2 deficient cells were hypersensitive to HU, whereas FANCD2 mono-ubiquitination deficient mutant cells, FANCD2 corrected cells, FANCA deficient cells and FANCJ deficient cells were not hypersensitive to HU. Our data indicate that FANCD2 plays an important role in PCNA mono-ubiquitination and translesion synthesis partially in a mono-ubiquitination independent manner. Rad51 also plays an important role in PCNA mono-ubiquitination and translesion synthesis in a HR independent fashion. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Timing matters: error-prone gap filling and translesion synthesis in immunoglobulin gene hypermutation

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2008.0197 ◽

2008 ◽

Vol 364 (1517) ◽

pp. 595-603 ◽

Cited By ~ 20

Author(s):

Julian E Sale ◽

Christopher Batters ◽

Charlotte E Edmunds ◽

Lara G Phillips ◽

Laura J Simpson ◽

...

Keyword(s):

Dna Damage ◽

Active Sites ◽

Somatic Hypermutation ◽

Translesion Synthesis ◽

Dna Lesions ◽

Immunoglobulin Gene ◽

Lesion Bypass ◽

Replication Fork Progression ◽

Activation Induced Deaminase ◽

Translesion Polymerases

By temporarily deferring the repair of DNA lesions encountered during replication, the bypass of DNA damage is critical to the ability of cells to withstand genomic insults. Damage bypass can be achieved either by recombinational mechanisms that are generally accurate or by a process called translesion synthesis. Translesion synthesis involves replacing the stalled replicative polymerase with one of a number of specialized DNA polymerases whose active sites are able to tolerate a distorted or damaged DNA template. While this property allows the translesion polymerases to synthesize across damaged bases, it does so with the trade-off of an increased mutation rate. The deployment of these enzymes must therefore be carefully regulated. In addition to their important role in general DNA damage tolerance and mutagenesis, the translesion polymerases play a crucial role in converting the products of activation induced deaminase-catalysed cytidine deamination to mutations during immunoglobulin gene somatic hypermutation. In this paper, we specifically consider the control of translesion synthesis in the context of the timing of lesion bypass relative to replication fork progression and arrest at sites of DNA damage. We then examine how recent observations concerning the control of translesion synthesis might help refine our view of the mechanisms of immunoglobulin gene somatic hypermutation.

Download Full-text

The role of hRev7, the accessory subunit of hPolζ, in translesion synthesis past DNA damage induced by benzo[a]pyrene diol epoxide (BPDE)

BMC Cell Biology ◽

10.1186/1471-2121-11-97 ◽

2010 ◽

Vol 11 (1) ◽

pp. 97 ◽

Cited By ~ 5

Author(s):

Jessica A Neal ◽

Kathryn L Fletcher ◽

J Justin McCormick ◽

Veronica M Maher

Keyword(s):

Dna Damage ◽

Translesion Synthesis ◽

Diol Epoxide ◽

Accessory Subunit

Download Full-text

Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

Eukaryotic Cell ◽

10.1128/ec.00260-09 ◽

2009 ◽

Vol 9 (2) ◽

pp. 315-324 ◽

Cited By ~ 10

Author(s):

Kristina H. Schmidt ◽

Emilie B. Viebranz ◽

Lorena B. Harris ◽

Hamed Mirzaei-Souderjani ◽

Salahuddin Syed ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Genetic Interaction ◽

Chromosomal Rearrangements ◽

Point Mutations ◽

Normal Growth ◽

Dna Helicase ◽

Dna Damage Checkpoint ◽

Lesion Bypass

ABSTRACT Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1Δ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3Δ mutant and are essential for survival of rev3Δ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1Δ or rrm3Δ helicase mutations, whereas mph1Δ is hypostatic to sgs1Δ. Previously reported slow growth of mph1Δ srs2Δ cells is accompanied by G2/M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.

Download Full-text