Full Member, (Genetics)
Department of Cancer Biology
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo, NY 14263
Telephone: (716) 845-4462
Fax: (716) 845-5906
E-mail:David.Kowalski@RoswellPark.org

Professor, Cellular & Molecular Biology Program
Roswell Park Graduate Division
University at Buffalo

Description of Research

Faithful DNA replication and repair of DNA damage are required for genome stability and contribute to the health and survival of all organisms. Checkpoint pathways modulate DNA replication during normal cell cycles and during DNA damage repair, but little is known about the genetic and molecular determinants. The laboratory is studying determinants that regulate DNA replication in chromosomes. Such information is important for understanding pathways that maintain genome stability in healthy cells. Defects in these pathways can lead to the genome instability present in cancer cells. The laboratory also studies DNA replication in cells treated with chemotherapeutic drugs that damage DNA. Of interest are the genes that mediate drug sensitivity and their functions in DNA replication, checkpoints and other processes. Budding yeast (S. cerevisiae) is used as a model organism. Key proteins involved in DNA replication, checkpoints and other processes are conserved between yeast and humans. Yeast offers several advantages including ease of genetic manipulation and availability of mutations in known genes. Also, yeast DNA array technology permits genome-wide analysis of gene expression as well as gene function.

Research progress and plans

Certain replication origins in yeast chromosomes are normally silent and stall replication fork progression through the chromosome. However, this laboratory found that silent origins are competent for replication initiation and can be activated by deletion of adjacent early-firing origins. The findings suggested a role for replication timing: silent origins may be programmed to activate late in S phase and normally replicated passively by adjacent early origins. Some silent origins function in transcription silencing through ORC, the origin recognition complex required for replication initiation. An ORC mutation leads to activation of silent origins, consistent with a role for a late-programmed initiation complex at silent origins. Work in progress is investigating factors that control origin silencing, including replication timing, chromosome context and chromatin.

This laboratory has examined the effects of a DNA alkylating antitumor drug on yeast chromosome replication, focusing on the influence of mutations in checkpoint genes. The antitumor drug inhibits replication origin activity and stalls fork progression, selectively at a replication origin. Also, the drug, together with mutations in checkpoint kinase genes that regulate replication timing, mediates activation of replication origins that are normally silent. Such alterations in the normal replication pattern in a chromosome may contribute to chromosome instability. Consistent with this, novel replication intermediates suggesting DNA breaks were detected. The findings may prove informative for drug action in cancer cells since the checkpoint kinases examined have human homologues that are mutated in cancer.

Anticancer agent Ecteinascidin 743 (ET743) alkylates DNA and was found to inhibit replication origin activation of simian virus 40 DNA in cells and to induce formation of inactive replication intermediates with unusual DNA structures. Clinical trials have shown that ET743 is a promising new option for treatment of several types of sarcoma.

We have screened the collection of S. cerevisiae deletion strains to identify novel genes that mediate cellular resistance to cisplatin (Huang et al., 2005). Twenty of the genes identified had not been previously linked to cisplatin resistance and belong to distinct functional groups, including nucleotide metabolism, mRNA catabolism, RNA pol II-dependent gene regulation and vacuolar transport. Some deletion strains were cross-resistant to particular cytotoxic agents but sensitive to others. The sensitivity of certain resistant strains to other cytotoxic agents may point to particular drug combinations that can overcome resistance caused by inactivation of genes identified in our screen.

Earlier we discovered a novel cis-acting component, called a DNA unwinding element (DUE), in replication origins from several organisms. The low DNA helical stability of DUEs can be predicted from the DNA sequence using computer programs that we have developed. DUEs also function in other processes including the regulation of transcription. New programs that perform a combined analysis of specific DNA sequences and helical stability localized a DUE in the transcription regulatory of the human c-MYC proto-oncogene. The predicted region of minimal helical stability in the c-MYC gene overlapped a known site for a single-strand-specific DNA binding protein and an unwound DNA region identified in cells expressing c-MYC. The programs can be accessed freely through a Web server at: http://www.gsa.buffalo.edu/dna/dk/

Key Publications

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