The phi-XA protein plays a role in the site specific cleavage of the (+) strand of phi-X RFI DNA, the displacement reaction, a ligation reaction and a transfer reaction. These reactions require a 30 nucleotide conserved sequence found in all icosahedral phages. Alterations of this region at the 3'-end prevent binding, alterations around the cut site control endonuclease action and alteration at the 5'-end prevent the transfer reaction. Two tyrosine moieties in the phi-XZ protein can form a covalent linkage with the dAMP residue at nucleotide 4306; these polypeptides will be isolated and sequenced. The sites at which the phi-XA protein interacts with the conserved 30 nucleotide sequence will be determined by methylation protection and by DNase """"""""footprinting"""""""" experiments. RFI plasmids containing one conserved sequence and one altered sequence on the same strand will be constructed. The above reactions will be examined. The influence of phi-X174 prohead and the phi-XC and J proteins on the phi-XA catalyzed reactions will be studied. The role played by each protein involved in the in vitro replication of Ad DNA will be examined. The initiation reaction (pTP-dCMP complex) requires three Ad-encoded proteins, the pTP, the Ad pol and the Ad DBP plus one host protein nuclear factor I. This reaction requires two sequences present within the first 50 bp at each end of Ad DNA. One sequence lies between nucleotides 9-18, while the other is the factor I binding site (18-49). DNA synthesis will be studied in mutants in which the position of these two sites and their polarity will be varied. We plan to clone these proteins; monoclonal antibodies to nuclear factor I have been isolated and we plan to isolate the Ad Polpolymerase and the pTP protein by linking their cDNA to expression vectors. Replication of the displaced strand will be examined. Plasmids containing ori regions that are terminally redundant yield single-strands that form panhandle structures which are replicated. The replication of SV-40 DNA has been carried out in vitro. HeLa cell extracts plus SV-40 large T antigen support replication of plasmids containing the SV-40 origin sequence; no replication was observed with plasmids lacking the origin site. Intermediates in the reaction accumulate and migrate as complicated structures. These intermediates arise from the ori region and move in both directions. We are now in the process of purifying the proteins involved in the replication of SV-40 DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM034559-10
Application #
3484812
Study Section
Special Emphasis Panel (NSS)
Project Start
1984-07-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Li, Zhuo; Pan, Miao; Santangelo, Thomas J et al. (2011) A novel DNA nuclease is stimulated by association with the GINS complex. Nucleic Acids Res 39:6114-23
Fu, Yu V; Yardimci, Hasan; Long, David T et al. (2011) Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell 146:931-41
Ladner, Jane E; Pan, Miao; Hurwitz, Jerard et al. (2011) Crystal structures of two active proliferating cell nuclear antigens (PCNAs) encoded by Thermococcus kodakaraensis. Proc Natl Acad Sci U S A 108:2711-6
Vijayakumar, Sangeetha; Dziegielewska, Barbara; Levin, David S et al. (2009) Phosphorylation of human DNA ligase I regulates its interaction with replication factor C and its participation in DNA replication and DNA repair. Mol Cell Biol 29:2042-52
Farina, Andrea; Shin, Jae-Ho; Kim, Do-Hyung et al. (2008) Studies with the human cohesin establishment factor, ChlR1. Association of ChlR1 with Ctf18-RFC and Fen1. J Biol Chem 283:20925-36
Takahashi, Tatsuro S; Basu, Abhijit; Bermudez, Vladimir et al. (2008) Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts. Genes Dev 22:1894-905
Chang, Y Paul; Wang, Ganggang; Bermudez, Vladimir et al. (2007) Crystal structure of the GINS complex and functional insights into its role in DNA replication. Proc Natl Acad Sci U S A 104:12685-90
Song, Wei; Levin, David S; Varkey, Johnson et al. (2007) A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes. J Biol Chem 282:22721-30
Giordano-Coltart, Jennifer; Ying, Carol Y; Gautier, Jean et al. (2005) Studies of the properties of human origin recognition complex and its Walker A motif mutants. Proc Natl Acad Sci U S A 102:69-74
Kelman, Z (2000) DNA replication in the third domain (of life). Curr Protein Pept Sci 1:139-54

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