Document Type


Date of Degree

Spring 2010

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Wold, Marc S

First Committee Member

Elcock, Adrian

Second Committee Member

Demali, Kris A

Third Committee Member

Washington, M Todd

Fourth Committee Member

Weeks, Daniel L

Fifth Committee Member

Kohen, Amnon


Replication Protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism including, but not limited to, DNA replication, DNA repair and recombination. The `canonical' RPA is composed of three subunits (RPA1, RPA2, and RPA3). In addition to the three canonical subunits, there is a human homolog to the RPA2 subunit, termed RPA4, which can substitute for RPA2 in complex formation. The resulting RPA complex has been termed `alternative' RPA (aRPA). The normal function of aRPA is not known; however, previous studies have shown that it does not support S-phase progression in vivo. The goal of this thesis was to characterize the function of aRPA in DNA replication, DNA repair and recombination and profile its expression in human tissues.

The studies presented in this thesis show that the aRPA complex has solution and DNA binding properties indistinguishable from the canonical RPA complex as determined by gel mobility shift assays. However, aRPA was unable to support DNA replication and inhibited canonical RPA function in a cell-free simian virus 40 system. aRPA inhibited both initiation and elongation of DNA synthesis in the SV40 system. Two regions of RPA4, the putative L34 loop and the C-terminal winged helix domain, were responsible for inhibiting SV40 DNA replication.

The mechanism of SV40 DNA replication inhibition during initiation and elongation was characterized using assays for DNA polymerase α and DNA polymerase δ. aRPA was shown to have reduced interaction with DNA polymerase α and was not able to efficiently stimulate DNA synthesis by DNA polymerase α on aRPA coated single-stranded DNA. However, aRPA stimulated DNA synthesis by DNA polymerase δ in the presence of PCNA and RFC even though a reduced interaction was observed between aRPA and polymerase δ.

The role of aRPA in DNA repair was also investigated. aRPA interacted with both Rad52 and Rad51 but had a reduced interaction with Rad51. However, aRPA was still able to stimulate Rad51-dependent strand exchange. aRPA also supported the dual incision/excision reaction of nucleotide excision repair. aRPA was less efficient in nucleotide excision repair than canonical RPA and this reduction was attributed to reduced interactions with the repair factor XPA. In contrast, aRPA exhibited higher affinity for damaged DNA than canonical RPA.

The expression of RPA4 and RPA2 was determined by quantitative PCR in established cell lines, human normal tissues and human tumor tissue. RPA4 was shown to be expressed in all normal tissues examined but the level of expression was tissue specific. Additionally, RPA4 expression was decreased in all tumor tissues examined and was at the limit of detection in established cell lines. Taken together, the results presented in this thesis suggest that aRPA is a `non-proliferative' form of RPA that functions to maintain the genomic stability of non-dividing cells.


alternative Replication Protein A, aRPA, DNA repair, DNA replication, Replication Protein A, RPA4


xii, 200 pages


Includes bibliographical references (pages 184-200).


Copyright 2010 Aaron Charles Mason

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Biochemistry Commons