Cockayne syndrome B protein stimulates apurinic endonuclease 1 activity and protects against agents that introduce base excision repair intermediates

Abstract

The Cockayne syndrome B (CSB) protein—defective in a majority of patients suffering from the rare autosomal disorder CS—is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP–DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2′-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging

“…Recently, CSB has been shown to interact physically and/or functionally with more proteins involved in the BER pathway including poly(ADP-ribose) polymerase 1 (PARP-1) and APE1 (Wong et al, 2007). PARP-1 is an abundant nuclear DNA damage surveillance protein that can be characterized as a "molecular nick sensor".…”

“…CSB also interacts with APE1, stimulating the AP site incision activity of APE1 in an ATP independent manner (Wong et al, 2007). It was suggested that the CSB-APE1 interaction is most critical to regions of the genome where complex DNA structures are formed, such as during transcription or replication, or at sites of recombination, where the local, relative concentrations of these proteins may also be higher.…”

“…It was suggested that the CSB-APE1 interaction is most critical to regions of the genome where complex DNA structures are formed, such as during transcription or replication, or at sites of recombination, where the local, relative concentrations of these proteins may also be higher. Thus, in addition to coordination with DNA glycosylases in endogenous base damage repair and PARP-1 in DNA damage responses, the interaction with APE1 suggests a more general function of CSB in modulating BER processes (Wong et al, 2007).…”

“…Recently, CSB deficient transformed fibroblasts were found to be hypersensitive to HmdU, which generates cytotoxic BER intermediates/products after incorporation. Hence, it was suggested that CSB deficient cells are defective in the removal of these cytotoxic BER intermediates, likely AP sites and/or DNA SSBs (Wong et al, 2007). Paraquat is a widely used broad spectrum herbicide toxin.…”

“…Human fibroblasts defective in CSB are also hypersensitive to methyl methanesulfonate (MMS) treatment (Wong et al, 2007). MMS introduces BER substrates/intermediates.…”