Human Replication Protein A-Rad52-Single-Stranded DNA Complex: Stoichiometry and Evidence for Strand Transfer Regulation by Phosphorylation

被引：34

作者：

Deng, Xiaoyi ^{[1
,2
]}

Prakash, Aishwarya ^{[1
]}

Dhar, Kajari ^{[1
]}

Baia, Gilson S. ^{[1
]}

Kolar, Carol ^{[1
]}

Oakley, Greg G. ^{[3
]}

Borgstahl, Gloria E. O. ^{[1
]}

机构：

[1] Univ Nebraska Med Ctr, Eppley Inst Res Canc & Allied Dis, Omaha, NE 68198 USA

[2] Univ Nebraska Med Ctr, Dept Biochem & Mol Biol, Omaha, NE 68198 USA

[3] Univ Nebraska Med Ctr, Coll Dent, Lincoln, NE 68583 USA

来源：

BIOCHEMISTRY | 2009年 / 48卷 / 28期

关键词：

HUMAN RAD52 PROTEIN; MIDDLE SUBUNIT RPA32; BINDING-PROTEIN; HOMOLOGOUS RECOMBINATION; SACCHAROMYCES-CEREVISIAE; A PHOSPHORYLATION; BREAK REPAIR; INDUCED HYPERPHOSPHORYLATION; P34; SUBUNIT; HUMAN-CELLS;

D O I：

10.1021/bi900564k

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential in DNA metabolism and is phosphorylated in response to DNA-damaging agents. Rad52 and RPA participate in the repair of double-stranded DNA breaks (DSBs). It is known that human RPA and Rad52 form a complex, but the molecular mass, stoichiometry, and exact role of this complex in DSB repair are unclear. In this study, absolute molecular masses of individual proteins and complexes were measured in solution using analytical size-exclusion chromatography coupled with multiangle light scattering, the protein species present in each purified fraction were verified via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western analyses, and the presence of biotinylated ssDNA in the complexes was verified by chemiluminescence detection. Then, employing UV cross-linking, the protein partner holding the ssDNA was identified. These data show that phosphorylated RPA promoted formation of a complex with monomeric Rad52 and caused the transfer of ssDNA from RPA to Rad52. This suggests that RPA phosphorylation may regulate the first steps or DSB repair and is necessary for the mediator function of Rad52.

引用

页码：6633 / 6643

页数：11

共 78 条

[1] Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair [J].

Anantha, Rachel W. ;

Vassin, Vitaly M. ;

Borowiec, James A. .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2007, 282 (49) :35910-35923

[2] Heteroduplex formation by human Rad51 protein: Effects of DNA end-structure, hRP-A and hRad52 [J].

Baumann, P ;

West, SC .

JOURNAL OF MOLECULAR BIOLOGY, 1999, 291 (02) :363-374

[3] RMPs: Recombination/replication mediator proteins [J].

Beernink, HTH ;

Morrical, SW .

TRENDS IN BIOCHEMICAL SCIENCES, 1999, 24 (10) :385-389

[4] Synergistic actions of Rad51 and Rad52 in recombination and DNA repair [J].

Benson, FE ;

Baumann, P ;

West, SC .

NATURE, 1998, 391 (6665) :401-404

[5] Replication Protein A phosphorylation and the cellular response to DNA damage [J].

Binz, SK ;

Sheehan, AM ;

Wold, MS .

DNA REPAIR, 2004, 3 (8-9) :1015-1024

[6]

Blackwell LJ, 1996, MOL CELL BIOL, V16, P4798

[7] Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21 [J].

Block, WD ;

Yu, YP ;

Lees-Miller, SP .

NUCLEIC ACIDS RESEARCH, 2004, 32 (03) :997-1005

[8] UV LIGHT-INDUCED DNA-SYNTHESIS ARREST IN HELA-CELLS IS ASSOCIATED WITH CHANGES IN PHOSPHORYLATION OF HUMAN SINGLE-STRANDED DNA-BINDING PROTEIN [J].

CARTY, MP ;

ZERNIKKOBAK, M ;

MCGRATH, S ;

DIXON, K .

EMBO JOURNAL, 1994, 13 (09) :2114-2123

[9]

D'Souza JS, 2006, CURR SCI INDIA, V91, P1641

[10] Stability and nuclear distribution of mammalian replication protein A heterotrimeric complex [J].

Dimitrova, DS ;

Gilbert, DM .

EXPERIMENTAL CELL RESEARCH, 2000, 254 (02) :321-327

← 1 2 3 4 5 6 7 8 →