scholarly journals Phosphorylation of Human DNA Ligase I Regulates Its Interaction with Replication Factor C and Its Participation in DNA Replication and DNA Repair

2009 ◽  
Vol 29 (8) ◽  
pp. 2042-2052 ◽  
Author(s):  
Sangeetha Vijayakumar ◽  
Barbara Dziegielewska ◽  
David S. Levin ◽  
Wei Song ◽  
Jinhu Yin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Nuclear Antigen ◽  
Dna Ligase ◽  
Replication Factor C ◽  
Phase Form ◽  
Replication Factor ◽  
Human Dna ◽  
Dna Ligase I ◽  
Factor C

ABSTRACT Human DNA ligase I (hLigI) participates in DNA replication and excision repair via an interaction with proliferating cell nuclear antigen (PCNA), a DNA sliding clamp. In addition, hLigI interacts with and is inhibited by replication factor C (RFC), the clamp loader complex that loads PCNA onto DNA. Here we show that a mutant version of hLigI, which mimics the hyperphosphorylated M-phase form of hLigI, does not interact with and is not inhibited by RFC, demonstrating that inhibition of ligation is dependent upon the interaction between hLigI and RFC. To examine the biological relevance of hLigI phosphorylation, we isolated derivatives of the hLigI-deficient cell line 46BR.1G1 that stably express mutant versions of hLigI in which four serine residues phosphorylated in vivo were replaced with either alanine or aspartic acid. The cell lines expressing the phosphorylation site mutants of hLigI exhibited a dramatic reduction in proliferation and DNA synthesis and were also hypersensitive to DNA damage. The dominant-negative effects of the hLigI phosphomutants on replication and repair are due to the activation of cellular senescence, presumably because of DNA damage arising from replication abnormalities. Thus, appropriate phosphorylation of hLigI is critical for its participation in DNA replication and repair.

scholarly journals Phosphorylation of Serine 51 Regulates the Interaction of Human DNA Ligase I with Replication Factor C and Its Participation in DNA Replication and Repair

2012 ◽  
Vol 287 (44) ◽  
pp. 36711-36719 ◽  
Author(s):  
Zhimin Peng ◽  
Zhongping Liao ◽  
Barbara Dziegielewska ◽  
Yoshi Matsumoto ◽  
Stefani Thomas ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Ligase ◽  
Replication Factor C ◽  
Replication Factor ◽  
Human Dna ◽  
Dna Ligase I ◽  
Dna Replication And Repair ◽  
Factor C

scholarly journals The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome

2015 ◽  
Vol 290 (34) ◽  
pp. 20919-20933 ◽  
Author(s):  
Pavana M. Hegde ◽  
Arijit Dutta ◽  
Shiladitya Sengupta ◽  
Joy Mitra ◽  
Sanjay Adhikari ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Dna Glycosylase ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Replication Factor ◽  
Terminal Domain ◽  
Human Dna ◽  
Factor C

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

scholarly journals Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

1999 ◽  
Vol 19 (2) ◽  
pp. 1038-1048 ◽  
Author(s):  
Hiroyuki Tanaka ◽  
Koichi Tanaka ◽  
Hiroshi Murakami ◽  
Hiroto Okayama
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Proliferating Cell Nuclear Antigen ◽  
Large Subunit ◽  
Nuclear Antigen ◽  
Temperature Sensitive ◽  
Replication Factor C ◽  
Replication Factor ◽  
Factor C ◽  
Proliferating Cell

ABSTRACT At the nonpermissive temperature the fission yeastcdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. Thecdc24 + gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1 +, rfc1 + (a fission yeast homologue of RFC1), andhhp1 +, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition,cdc24 + genetically interacts with the gene encoding the catalytic subunit of DNA polymerase ɛ, which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.

scholarly journals The Large Subunit of Replication Factor C (Rfclp/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Michael A McAlear ◽  
K Michelle Tuffo ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Dna Replication ◽  
Uv Radiation ◽  
Large Subunit ◽  
Restrictive Temperature ◽  
Replication Factor C ◽  
Replication Factor ◽  
Factor C

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

scholarly journals Mechanical Link between Cohesion Establishment and DNA Replication: Ctf7p/Eco1p, a Cohesion Establishment Factor, Associates with Three Different Replication Factor C Complexes

2003 ◽  
Vol 23 (8) ◽  
pp. 2999-3007 ◽  
Author(s):  
Margaret A. Kenna ◽  
Robert V. Skibbens
Keyword(s):  
Dna Replication ◽  
Replication Factor C ◽  
Biologically Relevant ◽  
Replication Factor ◽  
Chromatid Cohesion ◽  
Physical Linkage ◽  
Factor C ◽  
Redundant Functions ◽  
Mutant Cells

ABSTRACT CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled—a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.

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