Chl1 and Ctf4 are required for damage-induced recombinations

doi:10.1016/j.bbrc.2006.12.185

Biochemical and Biophysical Research Communications

Volume 354, Issue 1, 2 March 2007, Pages 222-226

https://doi.org/10.1016/j.bbrc.2006.12.185 Get rights and content

Abstract

Deletion mutants of CHL1 or CTF4, which are required for sister chromatid cohesion, showed higher sensitivity to the DNA damaging agents methyl methanesulfonate (MMS), hydroxyurea (HU), phleomycin, and camptothecin, similar to the phenotype of mutants of RAD52, which is essential for recombination repair. The levels of Chl1 and Ctf4 associated with chromatin increased considerably after exposure of the cells to MMS and phleomycin. Although the activation of DNA damage checkpoint did not affected in chl1 and ctf4 mutants, the repair of damaged chromosome was inefficient, suggesting that Chl1 and Ctf4 act in DNA repair. In addition, MMS-induced sister chromatid recombination in haploid cells, and, more importantly, MMS-induced recombination between homologous chromosomes in diploid cells were impaired in these mutants. Our results suggest that Chl1 and Ctf4 are directly involved in homologous recombination repair rather than acting indirectly via the establishment of sister chromatid cohesion.

Section snippets

Materials and methods

Yeast strains. Construction of yeast strains are described in Appendix A (Supplemental Materials and methods). The yeast strains used in this study are listed in Supplementary Table S1.

Survival analysis following DNA damage. For survival analysis under continuous exposure to DNA damaging agents, 10-fold serial dilutions with distilled water of logarithmically growing cells (constructed as described above) were spotted onto YPAD plates or YPAD plates containing the indicated concentrations of

chl1, ctf4, and rad52 cells are sensitive to a spectrum of DNA damage

Cohesin is involved in the linkage of sister chromatid around damaged DNA [5], [6], suggesting that sister chromatid cohesion is important for DNA repair. Chl1 and Ctf4 are not included in the cohesin complex but are related to sister chromatid cohesion. To elucidate the repair function of cohesion in more detail, we investigated the relationship of Chl1 and Ctf4 to DNA repair. We first examined the sensitivity of chl1 and ctf4 cells to the DNA-damaging agents methyl methanesulfonate (MMS),

Acknowledgments

We thank M. Arisawa, P. Hieter, and J.E. Haber for plasmids or strains used in the study. We thank all members of the Enomoto lab for their support. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan.

References (22)

T.L. Orr-Weaver
The ties that bind: localization of the sister-chromatid cohesin complex on yeast chromosomes
Cell
(1999)
S. Gruber et al.
Chromosomal cohesin forms a ring
Cell
(2003)
C. Sjögren et al.
Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae
Curr. Biol.
(2001)
E. Unal et al.
DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain
Mol. Cell
(2004)
L. Ström et al.
Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair
Mol. Cell
(2004)
M.L. Mayer et al.
Identification of RFC (Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae
Mol. Cell
(2001)
L.C. Kadyk et al.
Sister chromatid are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae
Genetics
(1992)
J.S. Hanna et al.
Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion
Mol. Cell. Biol.
(2001)
R.V. Skibbens
Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion
Genetics
(2004)
S.L. Gerring et al.
The CHL1 (CTF1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G₂/M
EMBO J.
(1990)

Y. Hirota et al.

Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase

Nucleic Acids Res.

(2000)

Cited by (19)

The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome
2017, Journal of Biological Chemistry
DNA replication in eukaryotic cells is performed by a multiprotein complex called the replisome, which consists of helicases, polymerases, and adaptor molecules. Human acidic nucleoplasmic DNA-binding protein 1 (AND-1), also known as WD repeat and high mobility group (HMG)-box DNA-binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. Although structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (pol α) to the DNA helicase complex (Cdc45-MCM2–7-GINS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homotrimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex, mediated by unknown participants other than Go Ichi Ni San. In addition, we show that AND-1 binds to DNA in vitro, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.
Error-Free DNA Damage Tolerance and Sister Chromatid Proximity during DNA Replication Rely on the Polα/Primase/Ctf4 Complex
2015, Molecular Cell
Citation Excerpt :
This result highlights once again a differential contribution of cohesin and the Polα/Primase/Ctf4 complex toward DDT. A diverse molecular purpose of Polα/Primase and cohesin during canonical recombination can also be deduced from their contribution to the donor choice: in cohesin mutants, the inter-sister recombination events decrease in favor of events involving the homologous chromosome (Covo et al., 2010; Tittel-Elmer et al., 2012), whereas ctf4Δ causes a decrease in both sister- and inter-homologous recombination in conditions of DNA damage (Ogiwara et al., 2007). Altogether, these results reveal that, despite cohesin and ctf4Δ mutants showing similar phenotypes in cohesion and DDT, cohesin aids TS by maintaining the proximity of the recombination donor, while the Polα/Primase/Ctf4 complex must affect some fundamental activity or step during recombination-mediated damage bypass.
Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.
Roles of ChlR1 DNA helicase in replication recovery from DNA damage
2013, Experimental Cell Research
Citation Excerpt :
Considering that ChlR1 interacts with several replication fork proteins including PCNA, Fen1 and Timeless [24,29,34], it is straightforward to suggest that ChlR1's functions are required at the replication fork during S-phase. Consistently, cells deficient for ChlR1/Chl1 are also sensitive to MMC, MMS, and CPT, all of which cause DNA breaks during replication [5,26,30,31], further supporting a notion that ChlR1 is involved in DNA repair at the replication fork. Previous studies reported that ChlR1 enhances activity of Fen1, a critical enzyme for Okazaki fragment processing during lagging strand DNA synthesis [29].
The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion.
CHL-1 provides an essential function affecting cell proliferation and chromosome stability in Caenorhabditis elegans
2011, DNA Repair
Citation Excerpt :
While much is known about the functions of these three helicases in the family, less is known about the biological function of CHL-1. The CHL1 gene was first identified in yeast in a screen for mutants with decreased fidelity of chromosome transmission [17,18] and later shown to be required for sister chromatid cohesion [19–22] and the repair of methylmethane sulfonate-induced DNA lesions [23,24]. Impaired function in the mammalian ortholog ChlR1 (CHL1-related 1)/DDX11 (DEAD/H box polypeptide 11) caused defects in sister chromatid cohesion in mouse embryos [25] and human cell cultures [26].
A family of helicases that are important in maintaining genome stability is the iron–sulfur group. Members of this family include DOG-1/FANCJ, RTEL1, XPD and Chl1p/DDX11. In Caenorhabitis elegans, the predicted gene M03C11.2 has orthology to the CHL1 (Chromosome loss 1) gene in Saccharomyces cerevisiae and DDX11 (DEAD/H box polypeptide 11) in humans. In this paper, we show that the chl-1 gene in C. elegans is required for normal development and fertility. Mutants have lineage-independent cell proliferation defects that result in a Stu (sterile uncoordinated) phenotype, characterized by gonadal abnormalities and a reduced number of D motor neurons and seam cells. A chromosome stability defect is present in the germ cells, where an abnormal number of DAPI-staining chromosomes appear in diakinesis. CHL-1 function is required for the integrity of poly-guanine/poly-cytosine DNA in the absence of DOG-1/FANCJ: the loss of CHL-1 alone does not result in the deletion of G-tracts, but it does increase the number of deletions observed in the dog-1; chl-1 double mutant, indicating a role for CHL-1 during replication and repair. In addition, we observed that cohesin defects increased the number of deletions in the absence of DOG-1/FANCJ. Our results demonstrate a role for CHL-1 in cell proliferation and maintaining normal chromosome numbers, and implicate CHL-1 in chromosome stability and repair of unresolved secondary structures during replication.
Cul8/Rtt101 forms a variety of protein complexes that regulate DNA damage response and transcriptional silencing
2010, Journal of Biological Chemistry
The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical analysis of Cul8 complexes. Cul8 was found to form a Cul8-Mms1-Mms22-Esc4 complex under physiological conditions, with Mms1 bridging Cul8 and Mms22 and Mms22 bridging Mms1 and Esc4. Domain analysis demonstrated that the N-terminal region of Mms1 and the C-terminal region of Mms22 are required for the Mms1-Mms22 interaction, whereas the N-terminal region of Mms22 is required for the Mms22-Esc4 interaction. We also found other Cul8-Mms1-binding proteins Ctf4, Esc2, and Orc5 using yeast two-hybrid screening. Esc4 and Ctf4 bound to Mms22 directly and bound to Cul8-Mms1 in the presence of Mms22, whereas Esc2 and Orc5 interacted with both Cul8 and Mms1, independently. We found that Cul8, Mms1, and Mms22 participated in the regulation of transcriptional silencing of yeast telomeres. These results suggest that Cul8-Mms1, as part of various protein complexes, is involved in the regulation of chromatin metabolism.
Roles of human AND-1 in chromosome transactions in S phase
2009, Journal of Biological Chemistry
Coordinated execution of DNA replication, checkpoint activation, and postreplicative chromatid cohesion is intimately related to the replication fork machinery. Human AND-1/chromosome transmission fidelity 4 is localized adjacent to replication foci and is required for efficient DNA synthesis. In S phase, AND-1 is phosphorylated in response to replication arrest in a manner dependent on checkpoint kinase, ataxia telangiectasia-mutated, ataxia telangiectasia-mutated and Rad3-related protein, and Cdc7 kinase but not on Chk1. Depletion of AND-1 increases DNA damage, delays progression of S phase, leads to accumulation of late S and/or G₂ phase cells, and induces cell death in cancer cells. It also elevated UV-radioresistant DNA synthesis and caused premature recovery of replication after hydroxyurea arrest, indicating that lack of AND-1 compromises checkpoint activation. This may be partly due to the decreased levels of Chk1 protein in AND-1-depleted cells. Furthermore, AND-1 interacts with cohesin proteins Smc1, Smc3, and Rad21/Scc1, consistent with proposed roles of yeast counterparts of AND-1 in sister chromatid cohesion. Depletion of AND-1 leads to significant inhibition of homologous recombination repair of an I-SceI-driven double strand break. Based on these data, we propose that AND-1 coordinates multiple cellular events in S phase and G₂ phase, such as DNA replication, checkpoint activation, sister chromatid cohesion, and DNA damage repair, thus playing a pivotal role in maintenance of genome integrity.

View all citing articles on Scopus

View full text

Article preview

Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

chl1, ctf4, and rad52 cells are sensitive to a spectrum of DNA damage

Acknowledgments

References (22)

The ties that bind: localization of the sister-chromatid cohesin complex on yeast chromosomes

Cell

Chromosomal cohesin forms a ring

Cell

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae

Curr. Biol.

DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain

Mol. Cell

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Mol. Cell

Identification of RFC (Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae

Mol. Cell

Sister chromatid are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae

Genetics

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

Mol. Cell. Biol.

Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion

Genetics

The CHL1 (CTF1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G₂/M

EMBO J.

Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase

Nucleic Acids Res.

Cited by (19)

The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome

Error-Free DNA Damage Tolerance and Sister Chromatid Proximity during DNA Replication Rely on the Polα/Primase/Ctf4 Complex

Roles of ChlR1 DNA helicase in replication recovery from DNA damage

CHL-1 provides an essential function affecting cell proliferation and chromosome stability in Caenorhabditis elegans

Cul8/Rtt101 forms a variety of protein complexes that regulate DNA damage response and transcriptional silencing

Roles of human AND-1 in chromosome transactions in S phase

Chl1 and Ctf4 are required for damage-induced recombinations

Abstract

Section snippets

Materials and methods

chl1, ctf4, and rad52 cells are sensitive to a spectrum of DNA damage

Acknowledgments

Cell

Cell

Curr. Biol.

Mol. Cell

Mol. Cell

Mol. Cell

Sister chromatid are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae

Genetics

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

Mol. Cell. Biol.

Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion

Genetics

The CHL1 (CTF1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M

EMBO J.

Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase

Nucleic Acids Res.

The CHL1 (CTF1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G₂/M