Author

Kevin McCabe

Date

7-2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Department of Molecular and Medical Genetics

Institution

Oregon Health & Science University

Abstract

The Fanconi anemia pathway aids in maintaining genomic stability through coordination of several repair pathways in order to respond to DNA interstrand crosslinking (ICL). The hererodimer ERCCI/XPF has been implicated in various contrasting roles in the repair ofiCL. Immunofluoresence and immunoblotting was carried out on lysates from ERCCI siRNA depleted GM639 human immortalized fibroblast cells. We demonstrate hERCCl is not required for the formation of double strand breaks in response to MMC, DEB, or HU, but is required for hFANCD2 monoubiquitination in response to these clastogens. Additionally, depletion ofhERCCl reduces the level of abnormal chromosome structures known as radials when co-depleted with hFANCA. We conclude hERCCl acts downstream ofH2AX phosphorylation, but upstream of the formation of radials and activation of the FA pathway, both in response to MMC, DEB and HU suggesting a common intermediate produced by these ICL inducing agents and HU induced stalled replication forks. Our work suggests the FA pathway acts not on double strand breaks, but rather, on an intermediate produced in the resolution of collapsed replication forks resulting from ICL inducing agents and stalled replication forks.

Identifier

doi:10.6083/M42V2D4K

School

School of Medicine

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