scholarly journals Genetic Interactions between the Escherichia coli umuDC Gene Products and the β Processivity Clamp of the Replicative DNA Polymerase

2001 ◽  
Vol 183 (9) ◽  
pp. 2897-2909 ◽  
Author(s):  
Mark D. Sutton ◽  
Mary F. Farrow ◽  
Briana M. Burton ◽  
Graham C. Walker
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Cold Sensitivity ◽  
Dna Damage Checkpoint ◽  
Gene Products ◽  
Missense Mutations ◽  
Dna Polymerase Iii ◽  
Checkpoint Control ◽  
Cold Sensitive ◽  
Growth Phenotype

ABSTRACT The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA–single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD′ (which enables TLS). In addition, even modest overexpression of theumuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD2C. We have previously reported that overexpression of the ɛ proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of ɛ with UmuD2C is important for the DNA damage checkpoint control function of theumuDC gene products. Here, we report that overexpression of the β processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of theumuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD′C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant β proteins possibly deficient for physical interactions with theumuDC gene products, we selected for noveldnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD′C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of β or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaNmissense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eightdnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of theumuDC gene products, suggesting that the interactions between UmuD′ and β are a subset of those between UmuD and β. Taken together, these findings suggest that interaction of β with UmuD2C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD2C with the ɛ and the β subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.

scholarly journals umuDC-Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD2C Complex Involved in a DNA Damage Checkpoint Control

2001 ◽  
Vol 183 (4) ◽  
pp. 1215-1224 ◽  
Author(s):  
Mark D. Sutton ◽  
Graham C. Walker
Keyword(s):  
Dna Damage ◽  
Polymerase Activity ◽  
Binding Activity ◽  
Cell Cycle Checkpoint ◽  
Cold Sensitivity ◽  
Gene Products ◽  
Checkpoint Control ◽  
Sos Mutagenesis ◽  
Dna Binding Activity ◽  
C Complex

ABSTRACT The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD′2C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show thatumuDC-mediated cold sensitivity can be genetically separated from the role of UmuD′2C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate thatumuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD2C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD′ in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of theumuDC gene products.

scholarly journals umuDC-dnaQ Interaction and Its Implications for Cell Cycle Regulation and SOS Mutagenesis inEscherichia coli

2001 ◽  
Vol 183 (3) ◽  
pp. 1085-1089 ◽  
Author(s):  
Mark D. Sutton ◽  
Sumati Murli ◽  
Timothy Opperman ◽  
Carly Klein ◽  
Graham C. Walker
Keyword(s):  
Dna Polymerase ◽  
Cell Cycle Regulation ◽  
Biological Activities ◽  
Cold Sensitivity ◽  
Specific Interactions ◽  
Gene Products ◽  
Checkpoint Control ◽  
Translesion Dna Synthesis ◽  
Pol Iii ◽  
Cycle Regulation

ABSTRACT The Escherichia coli SOS-regulated umuDCgene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD′ proteins, both encoded by the umuD gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of theumuDC gene products. Here we show that overproduction of the ɛ proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the umuDC gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD′ and the C-terminal domain of ɛ.

scholarly journals Schizosaccharomyces pombe Cells Lacking the Amino-Terminal Catalytic Domains of DNA Polymerase Epsilon Are Viable but Require the DNA Damage Checkpoint Control

2001 ◽  
Vol 21 (14) ◽  
pp. 4495-4504 ◽  
Author(s):  
Wenyi Feng ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control ◽  
Dna Polymerase Epsilon ◽  
Catalytic Domains ◽  
C Terminus ◽  
N Terminus ◽  
Polymerase Epsilon

ABSTRACT In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol ɛ) is encoded bycdc20 + and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol ɛ that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol ɛ (cdc20 Δ N-term ) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability ofcdc20 Δ N-term cells is dependent on expression ofrad3 +, hus1 +, andchk1 +, three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol ɛ, cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly forcdc20 Δ N-term cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol ɛ is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

DNA Damage Checkpoint Control of Mitosis in Fission Yeast

2000 ◽  
Vol 65 (0) ◽  
pp. 353-360 ◽  
Author(s):  
N. RHIND ◽  
B.A. BABER-FURNARI ◽  
A. LOPEZ-GIRONA ◽  
M.N. BODDY ◽  
J.-M. BRONDELLO ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control

scholarly journals Selective disruption of the DNA polymerase III  -  complex by the umuD gene products

2012 ◽  
Vol 40 (12) ◽  
pp. 5511-5522 ◽  
Author(s):  
M. C. Silva ◽  
P. Nevin ◽  
E. A. Ronayne ◽  
P. J. Beuning
Keyword(s):  
Dna Polymerase ◽  
Gene Products ◽  
Dna Polymerase Iii ◽  
Polymerase Iii

scholarly journals The Role of Dbf4/Drf1-Dependent Kinase Cdc7 in DNA-Damage Checkpoint Control

2008 ◽  
Vol 32 (6) ◽  
pp. 862-869 ◽  
Author(s):  
Toshiya Tsuji ◽  
Eric Lau ◽  
Gary G. Chiang ◽  
Wei Jiang
Keyword(s):  
Dna Damage ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control

scholarly journals Rfc5, in Cooperation with Rad24, Controls DNA Damage Checkpoints throughout the Cell Cycle inSaccharomyces cerevisiae

2000 ◽  
Vol 20 (16) ◽  
pp. 5888-5896 ◽  
Author(s):  
Takahiro Naiki ◽  
Toshiyasu Shimomura ◽  
Tae Kondo ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Damage ◽  
Consensus Sequence ◽  
Nucleoside Triphosphate ◽  
Dna Damage Checkpoint ◽  
Binding Motif ◽  
Loss Of Function ◽  
Checkpoint Control ◽  
Dna Damage Checkpoints ◽  
M Phase ◽  
Factor C

ABSTRACT RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Δ mutants are defective in all the G1-, S-, and G2/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine115 residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neitherrfc5-1 nor rad24-K115R single mutants are defective in the G1- and G2/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G1-, S- and G2/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.

scholarly journals DNA Polymerase η, the Product of the Xeroderma Pigmentosum Variant Gene and a Target of p53, Modulates the DNA Damage Checkpoint and p53 Activation

2006 ◽  
Vol 26 (4) ◽  
pp. 1398-1413 ◽  
Author(s):  
Gang Liu ◽  
Xinbin Chen
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Xeroderma Pigmentosum ◽  
Early Stage ◽  
Chemotherapeutic Agents ◽  
Dna Damage Checkpoint ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Independent Manner ◽  
P53 Activation

ABSTRACT DNA polymerase η (PolH) is the product of the xeroderma pigmentosum variant (XPV) gene and a well-characterized Y-family DNA polymerase for translesion synthesis. Cells derived from XPV patients are unable to faithfully bypass UV photoproducts and DNA adducts and thus acquire genetic mutations. Here, we found that PolH can be up-regulated by DNA breaks induced by ionizing radiation or chemotherapeutic agents, and knockdown of PolH gives cells resistance to apoptosis induced by DNA breaks in multiple cell lines and cell types in a p53-dependent manner. To explore the underlying mechanism, we examined p53 activation upon DNA breaks and found that p53 activation is impaired in PolH knockdown cells and PolH-null primary fibroblasts. Importantly, reconstitution of PolH into PolH knockdown cells restores p53 activation. Moreover, we provide evidence that, upon DNA breaks, PolH is partially colocalized with phosphorylated ATM at γ-H2AX foci and knockdown of PolH impairs ATM to phosphorylate Chk2 and p53. However, upon DNA damage by UV, PolH knockdown cells exhibit two opposing temporal responses: at the early stage, knockdown of PolH suppresses p53 activation and gives cells resistance to UV-induced apoptosis in a p53-dependent manner; at the late stage, knockdown of PolH suppresses DNA repair, leading to sustained activation of p53 and increased susceptibility to apoptosis in both a p53-dependent and a p53-independent manner. Taken together, we found that PolH has a novel role in the DNA damage checkpoint and that a p53 target can modulate the DNA damage response and subsequently regulate p53 activation.

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