dna repair 5 (2006) 479–490
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/dnarepair
Translesion DNA synthesis across non-DNA segments in
cultured human cells
Sheera Adar, Zvi Livneh
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Received 15 November 2005
Received in revised form 5 January
Accepted 9 January 2006
Published on line 13 February 2006
DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS),
carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the
TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches
of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both
the trimethylene and dodecamethylene inserts were bypassed with signiﬁcant efﬁciencies
in human cells, using both misinsertion and misalignment mechanisms. TLS across these
non-DNA segments was aphidicolin-sensitive, and did not require pol. In vitro primer
extension assays showed that puriﬁed pol, pol and pol were each capable of inserting
each of the four nucleotides opposite the trimethylene chain, but only pol and pol could
fully bypass it. Pol and pol, but not pol, could also insert each of the four nucleotides
opposite the dodecamethylene chain, but all three polymerases were severely blocked by
this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon
chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of
transient and local template-independent polymerase activity, and highlights the robust-
ness of the TLS system in human cells.
© 2006 Elsevier B.V. All rights reserved.
DNA repair mechanisms have evolved to efﬁciently cope with
a great variety of harmful agents constantly challenging the
integrity of the genome. Most powerful are the error-free DNA
repair mechanisms that eliminate the damaged nucleotides,
and restore the original DNA sequence . However, often
replication commences prior to the completion of repair, and
the unrepaired lesions form a barrier to replication fork pro-
gression. Such circumstances result in recruitment of the DNA
damage tolerance mechanisms, either translesion DNA syn-
thesis (TLS) [1–5] or homologous recombinational repair (HRR)
[6–8]. The translesion mode of DNA synthesis is, for the most
part, conducted by specialized DNA polymerases. It appears
Corresponding author. Tel.: +972 8 934 3203; fax: +972 8 934 4169.
E-mail address: firstname.lastname@example.org (Z. Livneh).
Incumbent of The Maxwell Ellis Professorial Chair in Biomedical Research.
that the common denominator of these translesion DNA poly-
merases is a more spacious and ﬂexible active site [9–14],
which allows the bypass of even extremely bulky lesions, but
resulting in lower ﬁdelity of polymerization [5,15]. The multi-
plicity of TLS polymerases in mammalian cells serves, at least
in part, to endow the TLS system with DNA damage speci-
ﬁcity, as indicated by the ability of speciﬁc TLS polymerases
to bypass particular lesions in vivo, with higher efﬁciency and
higher accuracy than other polymerases, thus minimizing the
otherwise mutagenic effect of the lesions [16–21].
One of the interesting questions concerning TLS is the
range of the DNA lesions that it can overcome. It would have
been anticipated that a robust TLS system would be able to
replicate across a large variety of DNA lesions, and perhaps
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