Abstract
BackgroundGenome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. We investigated the dynamics of XRCC1 and PCNA that act as molecular loading platforms and play a central role in this coordination.ResultsLocal DNA damage was introduced by laser microirradation and the recruitment of fluorescent XRCC1 and PCNA fusion proteins was monitored by live cell microscopy. We found an immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Fluorescence bleaching experiments (FRAP and FLIP) revealed a stable association of PCNA with DNA repair sites, contrasting the high turnover of XRCC1. When cells were repeatedly challenged with multiple DNA lesions we observed a gradual depletion of the nuclear pool of PCNA, while XRCC1 dynamically redistributed even to lesions inflicted last.ConclusionThese results show that PCNA and XRCC1 have distinct kinetic properties with functional consequences for their capacity to respond to successive DNA damage events.
Highlights
Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions
XRCC1 is less tightly associated with repair sites than PCNA XRCC1 and PCNA have no known enzymatic function, are present at repair sites and interact with a high number of different proteins suggesting that they act as loading platforms in DNA repair
Microirradiated sites stained positive for phosphorylated histone variant H2AX, a marker for double strand breaks (DSBs), and poly(ADP-Ribose) which is generated by PARP at single strand breaks (SSBs) (Additional file 1)
Summary
Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. Mammalian cells have to deal with a wide variety of different DNA lesions caused by cellular metabolites, replication errors, spontaneous disintegration and environmental influences These lesions can occur at successive times and in distant parts of the genome constituting a permanent threat to the genetic integrity. The repair of DNA lesions involves multiple steps including initial damage recognition, intracellular signaling and the recruitment of repair factors For the latter step so called loading platforms are considered to play a central role by locally concentrating and coordinating repair factors at sites of DNA damage. These loading platforms have no enzymatic activity of their own but interact with numerous proteins through highly conserved binding motifs. XRCC1 (X-ray cross complementing factor 1) and PCNA (proliferating cell nuclear antigen) both fulfill these criteria and are considered to act as central loading platforms in DNA replication and repair (reviewed in [4,5,6])
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