The Regulatory Interactions of p21 and PCNA in Human Breast Cancer
Annual rept. 1 Jul 1999-30 Jun 2000
MARYLAND UNIV BALTIMORE
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To better understand the role of DNA replication in breast cancer, it is essential to examine the machinery that carries out the DNA synthetic process. Our laboratory has successfully purified a complex of proteins from breast cells that is fully competent to carry out T-antigen dependent, SV4O origin specific DNA replication in vitro, which we have termed the DNA synthesome. Analysis of the constituent proteins of the DNA synthesome of malignant and non-malignant breast cells by two-dimensional polyacrylamide gel electrophoresis 2D PAGE has uncovered a modification in an essential DNA replication protein, proliferating cell nuclear antigen PCNA. 2D PAGE analysis revealed that PCNA of malignant breast cells resolves as two distinct species, an unmodified form and a modified form, while PCNA present in non-malignant cell resolves exclusively as one form, the modified form. PCNA functions by forming a trimeric sliding clamp that encircles the DNA and interacts with the DNA polymerases delta and epsilon. Polymerase delta carries out leading strand DNA synthesis, and although a role for polymerase E has not1 yet been ascribed, it has been hypothesized to function in DNA repair. Another protein that interacts with PCNA is p21WAFlCIPlSDI1. P21 is a CDK inhibitor that, when induced by p53 in response to DNA damage, binds PCNA and effectively competes away polymerase 8 leading to the efficient inhibition of DNA replication. This inhibition impedes the replication of damaged DNA and theoretically allots time for the cell to repair its damaged DNA. Therefore, any alterations of the PCNA molecule could potentially abrogate p2 1 binding leading to replication of damaged DNA andor insufficient time for DNA repair. It is our goal to study the interaction of p2 1 with the modified and non-modified forms of PCNA and to investigate any functional consequences alterations in PCNAIp2 1 binding may have on DNA replication, DNA repair, and DNA replication fidelity.
- Anatomy and Physiology
- Medicine and Medical Research
- Polymer Chemistry