Direct and indirect roles of RECQL4 in modulating base excision repair capacity

Abstract

RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Bloom and Werner's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 siRNA knockdown human fibroblasts accumulate more H(2)O(2)-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H(2)O(2)-induced DNA damage. RTS primary fibroblasts also accumulate more XRCC1 foci than control cells in response to endogenous or induced oxidative stress and have a high basal level of endogenous formamidopyrimidines. In cells treated with H(2)O(2), RECQL4 co-localizes with APE1, and FEN1, key participants in base excision repair. Biochemical experiments indicate that RECQL4 specifically stimulates the apurinic endonuclease activity of APE1, the DNA strand displacement activity of DNA polymerase beta, and incision of a 1- or 10-nucleotide flap DNA substrate by Flap Endonuclease I. Additionally, RTS cells display an upregulation of BER pathway genes and fail to respond like normal cells to oxidative stress. The data herein support a model in which RECQL4 regulates both directly and indirectly base excision repair capacity.

Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction

“…Oxidative Stress and UV Exposure Increases Nuclear RPS3-RECQL4 Interaction in a Dose-dependent Manner-The helicase domain of RECQL4 has been shown to be important for various DNA repair functions (13)(14)(15), and this domain is frequently mutated in RTS, Baller-Gerald Syndrome, and RAPA-DILLINO syndrome patients (23). RTS patients are more prone to osteoskeletal deformities and osteosarcoma (23), and some studies have shown them to have increased sensitivity to UV (15) and oxidative stress (14,33).…”

“…RTS patients are more prone to osteoskeletal deformities and osteosarcoma (23), and some studies have shown them to have increased sensitivity to UV (15) and oxidative stress (14,33). RPS3 is also important for the progression of osteosarcoma (28).…”

“…The human AP endonuclease APE1 reportedly inhibits helicase activity of WRN in similar way (39). Second, RECQL4 has been reported to stimulate enzymatic activity of base excision repair proteins like APE1, FEN1, and DNA polymerase ␤ (14). RPS3 can compete with APE1 to occupy AP site substrates.…”

“…The role of RECQL4 in replication initiation process has been well characterized by assessing some of its important interaction partners such as MCM10 and TOPBP1 (11,12). However, its role in repair processes like doublestranded break repair (13), base excision repair (14), and nucleotide excision repair (15) warrants more analysis. Characteriza-tion of its functional interactions with repair proteins will likely reveal its particular roles in these processes and delineate the contributions of RECQL4 as a signaling cofactor and as an active enzyme.…”

“…RTS patient cell lines and fibroblasts depleted of RECQL4 exhibit defects in the oxidative DNA damage repair (14). In contrast, knockdown of RPS3 enhances survival of cells treated with hydrogen peroxide (27).…”