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Defining the modular protein intereactions that coordinate recruitment of DNA polymerase alpha to initiate SV40 DNA replication
Author(s) -
Fanning Ellen,
Huang Hao,
Zhang Haijiang,
Weiner Brian E.,
Chazin Walter J.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.196.2
Subject(s) - helicase , dna replication , replication protein a , primase , dna polymerase , microbiology and biotechnology , eukaryotic dna replication , polymerase , biology , dna polymerase delta , replication factor c , dna polymerase ii , control of chromosome duplication , dna , genetics , dna binding protein , gene , polymerase chain reaction , transcription factor , rna , reverse transcriptase
SV40 DNA replication, a simple but powerful model system, can be reconstituted with purified recombinant proteins in vitro, providing an opportunity to identify at the atomic level how a small number of eukaryotic proteins interact to initiate replication at a defined origin. Indeed four recombinant proteins suffice to initiate replication. Previous work from our labs and others suggests that origin melting by the viral helicase T antigen (Tag) leads to a transient ternary complex of Tag, RPA, and ssDNA. This complex dissociates as more ssDNA emerges from the helicase, generating RPA‐ssDNA parental template. Tag associated with DNA polymerase alpha‐primase (pol‐prim) is proposed to then remodel the bound RPA into a weaker binding mode, allowing pol‐prim to gain access to the exposed ssDNA and begin primer synthesis (EMBO J 25 , 5516; NSMB 12 , 332). This working model predicts that specific contacts of pol‐prim with the helicase domain of Tag are required to initiate replication. To further test the model, we determined the solution structure of the Tag‐interacting domain of pol‐prim regulatory subunit p68, characterized the binding surfaces of p68 and Tag, and will present evidence that interaction between these surfaces is needed for initiation in a reconstituted system. A model in which multiple defined protein interactions coordinate initiation of replication will be presented. (NIH GM52948 to EF and GM65484 to WJC)