Polynucleotides encoding RPA4/RPA32 hybrid polypeptides

#The field of the invention relates to replication proteins and variants thereof which may be utilized in methods for assessing and modulating cell proliferation. In particular, the field of the invention relates to replication protein A 32 (“RPA32” otherwise referred to as “RPA2”), replication protein 4 (“RPA4”), variants thereof, polynucleotides encoding the polypeptides, and antisense polynucleotides thereof which may be utilized in methods for assessing and modulating cell proliferation.

#The human replication protein A (RPA) is a trimeric complex found not only in human cells but also in other mammalian species and lower eukaryotes. RPA is a single-stranded DNA (ssDNA)-binding protein essential for DNA replication, repair, recombination, and monitoring cellular DNA-damage at cell cycle checkpoints. The three peptides of human RPA have molecular masses of approximately 70 kD (for the polypeptide called “RPA70” or alternatively “RPA1”); 32 kD (for the polypeptide called “RPA32” or alternatively “RPA2”); and 14 kD (for the polypeptide called “RPA14” or alternatively “RPA3”). All three polypeptides have been cloned and their encoding sequences have been reported (Erdile et al., 1991, J. BIOL. CHEM. 266:12090-12098; Erdile et al., 1990, J. Biol. Chem. 265:3177-3182; and Umbricht et al., 1993, J. BIOL. CHEM. 268:6131-6138).

#RPA has been observed to exhibit functional activities associated with several characterized domains of the three RPA polypeptides. For example, RPA has been observed to bind ssDNA via several identified DNA-binding domains (DBDs) in the three polypeptides. RPA70 contains four domains: an N-terminal DBD-F domain and DNA binding domains (DBDs) A, B, and C. RPA32 contains three domains, including an N-terminal phosphorylation domain, a central DNA-binding domain (DBD-D), and a C-terminal winged-helix domain involved in protein-protein interactions. RPA14 includes a single domain referred to as DBD-E. Not all of the domains referred to as “DBDs” are involved directly in binding ssDNA. The N-terminal DBD-F domain of RPA70 is implicated in protein-protein interactions in DNA metabolism. The DBD-E domain (i.e., RPA14) has been shown to be structurally important for the formation of the RPA trimer.

#FIG. 1 schematically illustrates the structure of the RPA70 polypeptide (i.e., “RPA1”); the RPA32 polypeptide (i.e., “RPA2”); and the RPA14 polypeptide (i.e., “RPA3”).

#FIG. 2 schematically illustrates the structure of the RPA32 polypeptide (i.e., “RPA2”); and the RPA4 polypeptide.

#FIG. 3 illustrates cell cycle phenotype after RPA32 knockdown and RPA32 expression. As described in the Examples, HeLa cells were transfected with small interfering RNA (“siRNA”) for RPA32 (“si32”) and optionally transfected with RPA32 plasmid DNA. The cell cycle phenotype was assessed by flow cytometry.

#The present invention is described herein using several definitions, as set forth below and throughout the application.

#As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≦10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

#As used herein, the terms “includes” and “including” shall have the same meaning as the terms “comprises” and “comprising.”