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Nucleic Acids Research, 2003, Vol. 31, No. 6 1725-1734
© 2003 Oxford University Press

RPA is an initiation factor for human chromosomal DNA replication

Dávid Szüts, Lisa Kitching, Christo Christov, Aidan Budd, Sew Peak-Chew1 and Torsten Krude

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK and 1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

*To whom correspondence should be addressed. Tel: +44 1223 330111; Fax: +44 01223 336676; Email: tk1{at}mole.bio.cam.ac.uk
Present address:
Aidan Budd, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity. To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei. We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system. Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors. We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA. The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication. Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro. RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro. These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.


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