Structural characterisation of transcription and replication through cisplatin lesioned DNA

Replication of the genome is strongly inhibited when high fidelity
DNA polymerases encounter unrepaired DNA lesions, which can not be
processed. The highly stringent active sites of these polymerases
are unable to accommodate damaged bases and therefore DNA lesions
block the replication fork progression. In order to overcome this
problem, cells have evolved mechanisms for either repairing the
damage, or synthesising past it with specially adapted
polymerasases. Eukaryotic DNA polymerase eta (Pol eta), belonging
to the Y-family of DNA polymerases, is outstanding in its ability
to replicate through a variety of highly distorting DNA lesions
such as cyclobutane pyrimidine dimers (CPDs), which are the main
UV-induced lesions. Also cisplatin induced 1,2-d(GpG) adducts
(Pt-GGs), which are formed in a typical cancer therapy with
cisplatin can be processed by Pol eta. The bypass of such
intrastrand crosslinks by high fidelity DNA polymerases is
particularly difficult because two adjacent coding bases are
simultaneously damaged. Thus, replication by Pol eta allows
organisms to survive exposure to sunlight or, in the case of
cisplatin, gives rise to resistances against cisplatin treatment.
Mutations in the human POLH gene, encoding Pol eta, causes the
variant form of xeroderma pigmentosum (XP V), characterized by the
failure to copy through CPDs. This leads to strongly increased UV
sensitivity and skin cancer predisposition. This thesis describes
mechanistic investigations of the translesion synthesis (TLS)
process by S. cerevisiae DNA Pol eta at atomic resolution, which
were undertaken in collaboration with the Hopfner group. To study
this process, cisplatin lesioned DNA had to be prepared first. Once
this technique was established, the catalytic fragment of Pol eta
was crystallized as ternary complex with incoming
2',3'-dideoxycytidine 5'-triphosphate (ddCTP) and an primer -
template DNA containing a site specific Pt-GG adduct. The first
obtained structure shows the ddCTP positioned in a loosely bound
conformation in the active site, hydrogen bonded to the templating
base. Realizing the importance of the 3’ hydroxy group for
positioning the NTP and the DNA correctly inside the polymerase,
the complex was crystallized again with a 2’-deoxynucleoside
5’-triphosphate (dNTP). To prevent nucleotidyl transfer, primer
strands which terminate at the 3’-end with a 2’,3’ dideoxy ribose
were prepared by reverse DNA synthesis and used for
cocrystallization. The resulting crystals diffracted typically to
3.1-3.3Å resolution at a synchrotron light source. A Pol eta
specific arginine (Arg73 in yeast Pol eta) was identified for its
importance to position the dNTP correctly in the active site and
was shown to be necessary for lesion bypass. In contrast to the
fixed preorientation of the dNTP in the active site, the damaged
DNA is bound flexibly in a rather open DNA binding cleft.
Nucleotidyl transfer requires a revolving of the DNA, energetically
driven by hydrogen bonding of the templating base to the dNTP. For
the 3’dG of the Pt-GG, this step is accomplished by bona fide
Watson-Crick base pairs to dCTP and is biochemically efficient and
accurate. In contrast, bypass of the 5’dG of the Pt-GG is less
efficient and promiscuous for dCTP and dATP. Structurally, this can
be attributed to misalignment of the templating 5’dG due to the
rigid Pt crosslink. In cooperation with the Cramer group the
structural reasons for the blockage of RNA Polymerase II (RNAP II)
by the cisplatin lesion were elucidated. Using structural as well
as biochemical methods it could be shown that stalling results from
a translocation barrier that prevents delivery of the lesion to the
active site. AMP misincorporation occurs at the barrier and also at
an abasic site, suggesting that it arises from nontemplated
synthesis according to an 'A-rule' known for DNA polymerases. RNAP
II can bypass a cisplatin lesion that is artificially placed beyond
the translocation barrier, even in the presence of a G A mismatch.
Thus, the barrier prevents transcriptional mutagenesis.