LABORATORY
OF GENOMIC INTEGRITY
|
Roger
Woodgate, PhD, Chief Scientists
within the Laboratory of Genomic Integrity (LGI) are trying to understand the
mechanisms by which mutations are introduced into damaged DNA. Recent studies
have shown that many of the proteins implicated in the mutagenic process are
low-fidelity DNA polymerases that can replicate past damaged DNA in a process
termed translesion DNA synthesis (TLS). The important role played by these
polymerases in mutagenesis and carcinogenesis is typified by human pol eta,
which can replicate past cis-syn thymine dimers both accurately and
efficiently. Humans with defects in pol eta are afflicted with the xeroderma
pigmentosum variant phenotype and exhibit sensitivity to ultraviolet light
and are prone to sunlight-induced skin cancers. Scientists
in the LGI previously identified and cloned a DinB homolog from the archaeon Sulfolobus
solfataricus P2, which is called DNA polymerase IV (Dpo4).
Characterization of the enzyme reveals that the protein possesses many
biochemical properties similar to eukaryotic members of the Y-family
polymerases, including a propensity to bypass certain DNA lesions such as a
thymine-thymine cyclobutane pyrimidine dimer. X-ray crystallography studies
of S. solfataricus Dpo4 in complexes with lesion-containing DNA and an
incoming nucleotide reveal that the active site of the enzyme is sufficiently
large to accommodate a large aromatic hydrocarbon DNA lesion, such as Benzo[alpha]pyrene
diol epoxide (BPDE), a metabolic product of carcinogens commonly found in
cigarette smoke and automobile exhaust fumes. Studies with human DNA
polymerase iota revealed that it physically interacts with the proliferating
cell nuclear antigen (PCNA) and that such an interaction stimulates the
processivity of pol iota in a template-dependent manner in vitro.
Mutations in either the pol iota PCNA-binding motif (PIP-box) or the
interdomain connector loop of PCNA diminished binding between pol iota and
PCNA and concomitantly reduced PCNA-dependent stimulation of pol iota
activity. Furthermore, unlike wild-type pol iota, the pol iota PIP-box mutant
failed to accumulate into replication foci after cellular DNA damage,
indicating that an interaction between pol iota and PCNA is essential for
foci formation. PCNA therefore acts both as a scaffold and a modulator of the
activities involved in replication and appears to recruit and coordinate
replicative and TLS-polymerases so as to ensure genome integrity. |