Werner syndrome (WS) is a rare autosomal recessive disorder that displays many of the clinical symptoms of normal aging at an early age. From the second decade of life onward, WS patients develop pathologies that resemble many of the traits of normal aging, such as osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and cancer. 1) WS is caused by mutation of a single gene that encodes a 1432-amino acid protein (WRN).2) WRN possesses multiple enzyme activities, including DNA-dependent ATPase, exonuclease, and DNA helicase activities. [3][4][5] Somatic cells derived from WS patients display subtle replication defects, genomic instability, and altered telomere dynamics, suggesting an important role of WRN in DNA metabolic pathways. [6][7][8][9] This is consistent with observations that a variety of proteins that interact with WRN are involved in DNA replication, repair, recombination, transcription, and maintenance of telomere structure.3) We identified a new protein that interacts with WRN and designated it originally Werner Helicase Interacting Protein (WHIP) 10) and renamed it later as WRN interacting protein 1 (WRNIP1). WRNIP1 belongs to the AAAϩ class of the ATPase family and is conserved from Escherichia (E.) coli to human.
10)Mutation in the gene encoding the budding yeast ortholog of WRNIP1, maintenance of genome stability 1 (MGS1) causes hyper recombination and early aging in yeast cells. 10,11) Genetic analyses using MGS1 mutants revealed that Mgs1p is required to prevent genome instability caused by replication arrest and is not involved in the repair of DNA lesions.12) In addition, overproduction of Mgs1p is lethal or very toxic when it is combined with mutations in the genes that encode proteins involved in DNA replication, such as replication protein A, replication factor C, proliferating cell nuclear antigen and DNA polymerase d (Pold).
13)Mgs1p physically and functionally interacts in vivo with budding yeast Pol31, the second subunit of Pold.14) Human WRNIP1 also interacts directly with human Pold and stimulates the DNA synthesizing activity of Pold by increasing initiation frequency of DNA synthesis from primers annealed on template DNA (template-primer DNA).15) In this context, it is of note that WRN interacts with Pold 16) and stimulates the DNA synthesizing activity of Pold by increasing the length of synthesized DNA.17) Since WRNIP1 interacts with WRN, 10,18) WRNIP1 and WRN may interact with templateprimer DNA in a cooperative fashion to regulate the activity of Pold. However, the biochemical relationships between WRNIP1 and WRN at the template-primer DNA remain elusive.In this study, the biochemical relationships between purified WRNIP1 and WRN on template-primer DNA were analyzed.
MATERIALS AND METHODS
Protein PreparationPreparation of baculovirus for the expression of human WRNIP1 was described previously.
15)High-5 cells infected with recombinant virus encoding FLAG-WRNIP1 were cultured for 3 d and collected by centrifugation. Cells were lysed with...