Replication Protein-A Mediates the Association of Calf Thymus DNA Polymerase  -DNA Primase Complex with Guanine-Rich DNA Sequence

ABSTRACT In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G1-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. TheARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element ofARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase α onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase ɛ only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.

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Error-free bypass of 2-hydroxyadenine by human DNA polymerase with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

Nucleic Acids Research ◽

10.1093/nar/gkm568 ◽

2007 ◽

Vol 35 (15) ◽

pp. 5173-5181 ◽

Cited By ~ 26

Author(s):

E. Crespan ◽

U. Hubscher ◽

G. Maga

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Nuclear Antigen ◽

Human Dna ◽

Proliferating Cell

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Role of Protein−Protein Interactions in the Function of Replication Protein A (RPA): RPA Modulates the Activity of DNA Polymerase α by Multiple Mechanisms

Biochemistry ◽

10.1021/bi970473r ◽

1997 ◽

Vol 36 (28) ◽

pp. 8443-8454 ◽

Cited By ~ 78

Author(s):

Katherine A. Braun ◽

Ye Lao ◽

Zhigang He ◽

C. James Ingles ◽

Marc S. Wold

Keyword(s):

Dna Polymerase ◽

Protein Interactions ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Protein Protein Interactions ◽

Dna Polymerase Α

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Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2108 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2108-2115 ◽

Cited By ~ 112

Author(s):

K L Collins ◽

T J Kelly

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Protein A ◽

Replication Protein A ◽

T Antigen ◽

Dna Templates ◽

Single Stranded Dna ◽

Dna Polymerase Alpha ◽

Primase Complex

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

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