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J. Biol. Chem., Vol. 276, Issue 13, 10168-10177, March 30, 2001
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,
,
,

From the The proliferating cell nuclear antigen
(PCNA) is a highly conserved protein required for the assembly of the
DNA polymerase delta (pol
Head and Neck Cancer Research Program, Guys,
King's, and St. Thomas' Dental Institute and the
** Division of Molecular and Medical Genetics, Pediatrics Research Unit,
Guy's Campus, King's College London, London SE1 9RT, United
Kingdom, the § Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724, and the
Imperial Cancer Research
Fund Skin Tumor Laboratory, the Royal London Hospital, London
E12AT, United Kingdom
) holoenzyme. Because PCNAs from
Saccharomyces cerevisiae and human do not complement each
other using in vitro or in vivo assays, hybrids
of the two proteins would help identify region(s) involved in the
assembly of the pol
holoenzyme. Two mutants of human PCNA, HU1
(D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared
by swapping corresponding regions between the two proteins. In
solution, all PCNA assembled into trimers, albeit to different extents.
These PCNA variants were tested for stimulation of pol
and in
vitro replication of M13 and SV40 DNA as well as to stimulate the
ATPase activity of replication factor C (RF-C). Our data suggest that
in addition to the interdomain connecting loop and C terminus, an
additional site in the N terminus is required for pol
interaction.
PCNA mutants and hybrids that stimulated pol
and RF-C were
deficient in M13 and SV40 DNA replication assays, indicating that
PCNA-induced pol
stimulation and RF-C-mediated loading are not
sufficient for coordinated DNA synthesis at a replication fork.

To whom correspondence should be addressed: Dept. of
Craniofacial Development, GKT Dental Institute, Floor 28, Guy's
Hospital, King's College London, London SE1 9RT, United
Kingdom. Tel.: 44-207-955-4992; E-mail:
ahmed.waseem@kcl.ac.uk.
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