Zhen-Qiang Pan

Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases

Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring‐shaped protein. This protein (‘sliding clamp’) encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage‐T4 systems, the processivity factors are ring‐shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer‐template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage.

INHIBITION OF NUCLEOTIDE EXCISION REPAIR BY THE CYCLIN-DEPENDENT KINASE INHIBITOR P21

p21, a p53-induced gene product that blocks cell cycle progression at the G phase, interacts with both cyclindependent kinases and proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor for DNA polymerases and and is required for both DNA replication and nucleotide excision repair. Previous studies have shown that p21 inhibits simian virus 40 (SV40) DNA replication in HeLa cell extracts by interacting with PCNA. In this report we show that p21 blocks nucleotide excision repair of DNA that has been damaged by either ultraviolet radiation or alkylating agents, and that this inhibition can be reversed following addition of PCNA. We have determined that p21 is more effective in blocking DNA resynthesis than in inhibiting the excision step. We further show that a peptide derived from the carboxyl terminus of p21, which specifically interacts with PCNA, inhibits polymerase -catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA. When added at higher levels, this peptide also blocks both SV40 DNA replication and nucleotide excision repair in HeLa cell extracts. These results indicate that p21 interferes with the function of PCNA in both in vitro DNA replication and nucleotide excision repair.

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2–q24.3, 3q27, 13q12.3–q13, and 7q11.23

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.