Chemistry Reference
In-Depth Information
tained a
32
P-labeled phosphate at the phosphodiester bond near the lesion on its 5
′
side. Following incubation with cell extract or a reconstituted system containing
highly purifi ed NER factors, damage-containing oligonucleotides are released as
radiolabelled DNA fragments that are visualized after gel electrophoresis and auto-
radiography. Incubation in mammalian cell extracts or a reconstituted system con-
taining highly purifi ed NER factors demonstrated that both major 1,2- and minor
1,3-intrastrand crosslinks of cisplatin are effi ciently repaired, the 1,3-crosslink being
repaired much more effi ciently.
61
Both crosslinks bend and unwind DNA to a similar
extent, but in contrast to 1,2-intrastrand crosslinks, the 1,3-intrastrand adduct of
cisplatin locally denatures and induces a fl exibility at the site of the 1,3-adduct.
44
Hence, the differential NER can be attributed to the different DNA structures
induced by the 1,2- and 1,3-intrastrand cisplatin adducts. Denatured base pairs and
locally enhanced fl exibility induced in DNA by platinum complexes may be factors
enhancing recognition of platinum adducts by NER systems. On the other hand, in
the extracts from human HeLa cells, the 1,2-AG intrastrand crosslink is excised
more effi ciently than the 1,2-GG intrastrand adduct
61
although these 1,2-intrastrand
crosslinks distort the DNA in a similar way (DNA unwinding and bending are
similar
6,99,100
and hydrogen bonds in the distorted base pairs are preserved).
NER of 1,2-, but not of 1,3-intrastrand crosslinks, is blocked upon addition of
an HMG - domain protein (HMG = high mobility group).
101
Several mechanisms
have been considered for how HMG-domain proteins might modulate the sensitiv-
ity of cells to cisplatin.
61
One prominent hypothesis is that HMG-domain proteins
block cisplatin-DNA adducts from the damage recognition needed for repair.
Hence, in the cells where this shielding mechanism is responsible for their sensitivity
to cisplatin, 1,2- and not 1,3-intrastrand crosslinks of this metallodrug are most likely
candidates for genotoxic lesions relevant to the antitumour effects of cisplatin.
An
in vitro
NER of a site-specifi c cisplatin interstrand crosslink has also been
studied using mammalian cell-free extracts containing HMG-domain proteins at
levels insuffi cient to block NER of the 1,2-intrastrand adducts.
61
Repair of the inter-
strand crosslink formed by cisplatin was not detected. Similarly, in cell strains
derived from patients with Fanconi's anaemia, NER of cisplatin-interstrand
crosslinks is not observed, although NER can readily occur in these cells.
102,103
Fan-
coni's anaemia cells have been described as being extremely sensitive to interstrand
crosslinking agents, and it was suggested that this high sensitivity to cisplatin could
be explained by the incapability of these cells to repair cisplatin interstrand
crosslinks.
104
On the other hand, repair of these lesions has been detected with the
aid of a repair synthesis assay, which measures the amount of new DNA synthesized
after damage removal in whole-cell extracts.
105
In this way, however, the repair could
also result from a mechanism different from that of NER. DNA interstrand crosslinks
pose a special challenge to repair enzymes because they involve both strands of
DNA and therefore cannot be repaired using the information in the complementary
strand for resynthesis. Quite recently the processing of stalled forks caused by DNA
interstrand crosslinks has been proposed to be an important step in initiating mam-
malian interstrand crosslink repair. It has been demonstrated
106
that the XPF-
ERCC1 complex makes an incision 5
to a psoralen interstrand crosslink on Y-shaped
DNA and that the XPF-ERCC1 complex generates an interstrand crosslink-specifi c
incision on the 3
′
′
-side of this lesion. The interstrand crosslink-specifi c 3
′
incision,
