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Abstract

Cloning and Sequencing of Partial cDNAs Derived from Canine DNA Repair Genes Linking Cancer and Aging

Tanya L. Gustafson and Keith E. Murphy*

Department of Pathobiology, Texas A&M, College of Veterinary Medicine, College Station, TX 77843-4467

Cancer is the most frequent disease of the domestic dog, Canis familiaris.  There are strong implications for causal relationships between increasing age, decreased DNA repair capacity, and development of cancer.  Also, assessment of expression of various DNA repair genes is used as a diagnostic tool and for prognosis in analysis of some human cancers.  However, DNA repair genes are largely uncharacterized the dog.  In this study, cDNA fragments derived from five canine DNA repair genes (ERCC2, MSH2, POLB, MRE11, and WRN), representing each of the five major repair pathways (nucleotide excision repair, mismatch repair, base excision repair, non-homologous end-joining and homologous recombinational repair), have been cloned and sequenced.

(Keywords: cancer, aging, DNA repair)

INTRODUCTION

   Chemical agents, irradiation and spontaneous enzymatic errors are constantly damaging DNA in all cells.  There are five DNA repair mechanisms that can be used after damage occurs, nucleotide excision repair (NER), mismatch repair (MMR), base excision repair (BER), non-homologous end-joining (NHEJ) and homologous recombinational repair (HRR).  The importance of DNA repair is reflected in the fact that proteins involved in each of these repair processes have roles in cancer and aging in the human. 

   Our interest is in the genetic basis of diseases affecting the domestic dog, Canis familiaris.  Because the dog is unique in the extent to which it shares our environment (and thus variables that influence cancer development and aging), it is ideal for a study designed to understand the genetics of cancer and aging.  Such work will be of benefit to the dog and human.  Importantly, the more than 400 dog breeds represent a slightly simplified model system because of their intra-breed genetic homogeneity.  At the same time, breeds ranging across a spectrum of cancer susceptibilities and lifespans are available for study due to selective breeding programs over the past hundreds of years.

   Excision repair cross-complementing 2 gene (ERCC2) encodes the XPD helicase, which is essential for NER and p53-mediated apoptosis.  Mutations in ERCC2 can cause different human diseases: two of which are xeroderma pigmentosum and Cockayne's syndrome.  Xeroderma pigmentosum is associated with increased risk of skin cancer, and Cockayne's syndrome is clinically similar to progeria.  At the same time, other mutations in ERCC2 can increase protection from lung cancer and basal cell carcinoma (Bernstein et al., 2002).   

   Msh2 protein forms part of the MutSa heterodimer, which detects errors occurring due to the limited fidelity of the polymerases replicating DNA or due to chemical damage to DNA.  Mutations in MSH2 cause approximately half of all cases of hereditary nonpolyposis colorectal cancer in an autosomal dominant pattern (Yehuda et al., 2000; Papadopoulos and Lindblom, 1997).  Evidence of declining mismatch repair with age was provided by Yehuda et al. (2000), who used microsatellite instability as an inverse measure of MMR capacity to demonstrate declining MMR capacity with age in humans.

   Polymerase b functions in BER to fill in single-stranded gaps in the DNA.  POLB expression decreases with age in several mouse tissues, but POLB is overexpressed in breast and colon adenocarcinomas (Cabelof et al., 2002; Srivastava et al., 1999). 

   X-ray-induced double-strand break repair capacity has been found to diminish with age in human peripheral blood lymphocytes (Mayer et al., 1989).  Mre11 forms a complex with Rad50 and Nbs1 to function in HRR and NHEJ, recognizing the DNA damage and processing the DNA double-strand breaks.  Mutations in human MRE11 lead to ataxia-telangiectasia-like disorder, a cancer predisposition syndrome.  Mre11 also has a role in maintenance of telomere length, linking it to cellular aging (Stewart et al., 1999; Connelly and Leach, 2002). 

   Werner's syndrome protein (WRN) is a RecQ helicase, which functions at the replication fork and promotes HRR under DNA damaging conditions.  Werner's syndrome is a rare autosomal recessive disease, resulting from mutational inactivation of WRN, characterized by premature aging and increased incidence of sarcomas (Yu et al., 1997; Bernstein et al., 2002). 

   It is interesting to postulate that decreased DNA repair capacity as dogs age is responsible for cancer susceptibility.  The first step in studying such a correlation is to characterize key DNA repair genes in the dog.  This paper reports the sequence of cDNA fragments derived from ERCC2, MSH2, POLB, MRE11 and WRN