Functional and Physical Interaction between WRN Helicase and Human Replication Protein A

Abstract

The human premature aging disorder Werner syndrome (WS) is associated with a large number of symptoms displayed in normal aging. The WRN gene product, a DNA helicase, has been previously shown to unwind short DNA duplexes (<53 base pairs) in a reaction stimulated by single-stranded DNA-binding proteins. We have studied the helicase activity of purified WRN protein on a variety of DNA duplex substrates to characterize the unwinding properties of the enzyme in greater detail. WRN helicase can catalyze unwinding of long duplex DNA substrates up to 849 base pairs in a reaction dependent on human replication protein A (hRPA). Escherichia coli SSB and bacteriophage T4 gene 32 protein (gp32) completely failed to stimulate WRN helicase to unwind long DNA duplexes indicating a specific functional interaction between WRN and hRPA. So far, there have been no reports of any physical interactions between WRN helicase and other proteins. In support of the functional interaction, we demonstrate a direct interaction between WRN and hRPA by coimmunoprecipitation of purified proteins. The physical and functional interaction between WRN and hRPA suggests that the two proteins may function together in vivo in a pathway of DNA metabolism such as replication, recombination, or repair.

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

“…Studies from yeast show that DNA replication does not proceed normally in absence of RecQ helicase function (Stewart et al, 1997) and in Xenopus laevis the ortholog of WRN is absolutely required for proper formation of replication foci (Yan et al, 1998). Functional interaction between proteins involved in DNA replication, such as replication protein A (RPA) and DNA polymerase ␦, and WRN also have been reported (Shen et al, 1998;Brosh et al, 1999;Kamath-Loeb et al, 2000). Furthermore, in yeast, the RecQlike proteins seem involved in suppression of hyperrecombination, S-phase checkpoint control, and correct DNA segregation (Gangloff et al, 1994, Watt et al, 1995Stewart et al, 1997;Davey et al, 1998;Yamagata et al, 1998;Frei and Gasser, 2000).…”

Deficient DNA repair in the human progeroid disorder, Werner syndrome

“…Recently it has been shown that WRN knockdown cells are hypersensitive to the methylating agents methyllexitropsin and temozolomide compared to isogenic controls [25]. In addition, WRN has been shown to physically interact with several proteins involved in SSBR/BER including: DNA polymerase ␦ (pol ␦) [26], DNA polymerase ␤ (pol ␤) [27], proliferating cell nuclear antigen (PCNA) [28], replication protein A (RPA) [29], flap endonuclease 1 (FEN-1) [30], and poly(ADPribose) polymerase 1 (PARP-1) [31]. WRN has been shown to stimulate FEN-1 flap cleavage [30] and nucleotide incorporation by pol ␦ [32].…”

“…WRN helicase activity stimulates pol ␤ strand displacement DNA synthesis [27] and cooperates with pol ␤ on 3 mismatches (Harrigan et al, unpublished). RPA stimulates WRN helicase activity [29] and the poly(ADP-ribosyl)ation state of PARP-1 regulates WRN helicase and exonuclease activities [33]. PARP-1 binds strongly to strand breaks and acts in the DNA damage surveillance network, partly by ribosylating a variety of nuclear proteins in response to DNA damage.…”

WRN promoter methylation possibly connects mucinous differentiation, microsatellite instability and CpG island methylator phenotype in colorectal cancer

“…3 Normal telomere function is an important cellular mechanism against aging and manifestations of Werner syndrome. [4][5][6] WRN can interact with p53, 7,8 and replication protein A1, 9 the latter of which is required for stabilization of single-stranded DNA during DNA replication. 10 Considering the importance of the WRN helicase in the maintenance of telomere function, WRN likely acts as a 'caretaker' tumor suppressor gene for genome integrity.…”