Functional interaction between the Werner Syndrome protein and DNA polymerase δ

Abstract

Werner Syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a 1,432-amino acid polypeptide (WRN) with a central domain homologous to the RecQ family of DNA helicases. Purified WRN unwinds DNA with 335 polarity, and also possesses 335 exonuclease activity. Elucidation of the physiologic function(s) of WRN may be aided by the identification of WRN-interacting proteins. We show here that WRN functionally interacts with DNA polymerase ␦ (pol ␦), a eukaryotic polymerase required for DNA replication and DNA repair. WRN increases the rate of nucleotide incorporation by pol ␦ in the absence of proliferating cell nuclear antigen (PCNA) but does not stimulate the activity of eukaryotic DNA polymerases ␣ or , or a variety of other DNA polymerases. Moreover, we show that functional interaction with WRN is mediated through the third subunit of pol ␦: i.e., Pol32p of Saccharomyces cerevisae, corresponding to the recently identified p66 subunit of human pol ␦. Absence of the third subunit abrogates stimulation by WRN, and stimulation is restored by reconstituting the three-subunit enzyme. Our findings suggest that WRN may facilitate pol ␦-mediated DNA replication and͞or DNA repair and that disruption of WRN-pol ␦ interaction in WS cells may contribute to the previously observed S-phase defects and͞or the unusual sensitivity to a limited number of DNA damaging agents. Werner Syndrome (WS) is an autosomal, recessive progeroid disorder characterized by genomic instability (1). Patients with WS prematurely exhibit age-related conditions such as atherosclerosis, osteoporosis, diabetes mellitus, and bilateral cataracts. Additionally, they show an increased incidence of cancers of non-epithelial cell lineage (2). Genetic instability in WS is manifested at the chromosomal level by breaks and rearrangements, and at the DNA level by multiple, large deletions (3-5).The WS gene encodes a 1,432-amino acid polypeptide (WRN) containing a domain homologous to the RecQ family of DNA helicases (6). This family is represented by the prototypical Escherichia coli RecQ (7), Saccharomyces cerevisiae Sgs1 (8), Schizosaccharomyces pombe Rqh1 (9), Xenopus laevis FFA-1 (Focus forming activity) (10), and the human proteins RecQL (11), BLM (the product of the Bloom's syndrome gene) (12), RecQ4 (the product of the Rothmund-Thomson syndrome gene) and RecQ5 (13,14).Several RecQ family members, including WRN, have been purified and shown to possess 3Ј35Ј DNA unwinding activity in vitro (15)(16)(17)(18)(19)(20). WRN helicase activity exhibits several features: First, unwinding of duplex DNA is ATP-dependent and typically requires a 3Ј single-stranded tail (19,21). Second, the activity is nonprocessive, limited to unwinding small stretches (Ͻ25 nt) of duplex DNA. Third, processivity is increased by ssDNA binding proteins, particularly by human replication protein A (22). In the presence of human replication protein A, WRN can unwind duplex DNA segments as long ...

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex

“…In contrast to the previous reports suggesting that WRN may function as a key factor in resolving aberrant DNA structures that arise from DNA metabolic processes Cheng et al, 1990;Constantinou et al, 2000;Fukuchi et al, 1989;Kamath-Loeb et al, 2000;Lebel et al, 1999;Salk et al, 1981), our study shows a novel function of WRN in accelerating rRNA transcription Figure 4 Pulse-chase analysis of rRNA synthesis in normal and WS ®broblasts. Normal ®broblasts (A/N0014) and WS ®broblasts (A/WS10801) were cultured at 378C for 48 h. Each culture was pulse-labeled with [ 3 H]-uridine (40 mCi/ml) for 2 h, and then was chased with 2 mM non-radioactive uridine for 1 h and 2 h. The radioactivity of total RNA prepared from each sample was counted.…”

“…Although Ku proteins have no e ect on the ATPase or helicase activity of WRN, they strongly stimulate speci®c exonuclease activity of WRN (Cooper et al, 2000). Kamath-Loeb et al (2000) reported that WRN stimulates DNA synthesis by DNA polymerase d, an essential eukaryotic polymerase with central roles in DNA replication and DNA repair. In addition, the murine homolog of WRN has been shown to be copuri®ed with the DNA replication complex and to interact with proliferating cell nuclear antigen (PCNA) and topoisomerase I (Lebel et al, 1999).…”

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

“…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).…”