Characterization of RTEL/PCNA interaction in the maintenance of genomic stability
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Date
2010-09-14T13:57:52Z
Authors
Nabi, Md. Zinnatun
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Abstract
Previously, we have demonstrated that a DNA helicase-like protein, termed RTEL (regulator of telomere length) is essential for the maintenance of genomic stability. RTEL deficiency induced telomere loss and genomic instability, leading to embryonic lethality. However, the role of RTEL in these biological pathways is largely unknown. To uncover RTEL’s function(s), we applied several approaches to identify the proteins that could interact with RTEL. Proliferating Cell Nuclear Antigen (PCNA), the key regulator of the replication fork, was found to be a strong candidate. In this study, we have demonstrated the interaction between RTEL and PCNA. Further characterization of the interaction between RTEL and PCNA revealed that the interaction is important for maintaining genomic stability. Due to the essential role of PCNA in nucleic acid metabolism as a component of the replication and repair machinery, its interaction with RTEL could be the key to the role of RTEL in the maintenance of genomic stability and mouse development. Along with a bioinformatics approach, we have employed several biochemical approaches to identify the interaction of PCNA with RTEL. Using co-immunoprecipitation, we have demonstrated that RTEL can specifically interact with PCNA. A PCR-based mutagenesis method was used to mutate the PCNA-interacting motif (PIP) in RTEL. Further we have demonstrated that several key amino acids in the PIP motif are responsible for mediating RTEL/PCNA interaction by using co-immunoprecipitation and immunofluorescence studies. Using a gene-targeting approach, we have specifically knocked-in a mutant RTEL with a mutation in PIP motif into mouse genome. Thus we have developed a transgenic mouse model to study the significance of the interaction between RTEL/PCNA in vivo. This study not only validated the interaction of RTEL with PCNA, via the PIP box, but also generated the RTEL PIP mutant alleles for further functional analysis by transgenic approaches. We have employed biochemical and cytogenetic studies to characterize the phenotypes in RtelI1169A/I1169A mouse. This is the first direct genetic approach to address whether PCNA is an important downstream mediator of RTEL’s function in the regulation of genomic integrity
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Molecular Biology, Transgenic model