Functions of alternative replication protein A in initiation and elongation.
Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair, and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative replication protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to those of the canonical RPA complex but does not support DNA replication. We describe studies that aimed to identify what properties of aRPA prevent it from functioning in DNA replication. We show aRPA has weakened interaction with DNA polymerase alpha (pol alpha) and that aRPA is not able to efficiently stimulate DNA synthesis by pol alpha on aRPA-coated DNA. Additionally, we show that aRPA is unable to support de novo priming by pol alpha. Because pol alpha activity is essential for both initiation and Okazaki strand synthesis, we conclude that the inability of aRPA to support pol alpha loading causes aRPA to be defective in DNA replication. We also show that aRPA stimulates synthesis by DNA polymerase alpha in the presence of PCNA and RFC. This indicates that aRPA can support extension of DNA strands by DNA polymerase partial differential. This finding along with the previous observation that aRPA supports early steps of nucleotide excision repair and recombination indicates that aRPA can support DNA repair synthesis that requires polymerase delta, PCNA, and RFC and support a role for aRPA in DNA repair.
Mason
Aaron C
AC
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Roy
Rupa
R
Simmons
Daniel T
DT
Wold
Marc S
MS
D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase