2 research outputs found
Development of Comprehensive Ultraperformance Liquid Chromatography–High-Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA–DNA Cross-Links
Cisplatin
(CP) is a common antitumor drug that is used
to treat
many solid tumors. The activity of CP is attributed to the formation
of DNA–DNA cross-links, which consist of 1,2-intra-, 1,3-intra-,
and interstrand cross-links. To better understand how each intrastrand
cross-link contributes to the activity of CP, we have developed comprehensive
ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM)
assays to quantify 1,2-GG-, 1,2-AG-, 1,3-GCG-, and 1,3-GTG-intrastrand
cross-links. The limit of quantitation for the developed assays ranged
from 5 to 50 fmol or as low as 6 cross-links per 108 nucleotides.
To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed
that the 1,2-GG-intrastrand cross-links were the most abundant intrastrand
cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand
cross-links. Furthermore, we investigated the repair kinetics of intrastrand
cross-links in CP-treated wild-type and nucleotide excision repair
(NER)-deficient U2OS cells. We observed a slow decrease of both 1,2-
and 1,3-intrastrand cross-links in wild-type cells and no evidence
of direct repair in the NER-deficient cells. Taken together, we have
demonstrated that our assays are capable of accurately quantifying
intrastrand cross-links in CP-treated samples and can be utilized
to better understand the activity of CP
Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome
Expansions of (CTG)·(CAG) repeated DNAs are the
mutagenic
cause of 14 neurological diseases, likely arising through the formation
and processing of slipped-strand DNAs. These transient intermediates
of repeat length mutations are formed by out-of-register mispairing
of repeat units on complementary strands. The three-way slipped-DNA
junction, at which the excess repeats slip out from the duplex, is
a poorly understood feature common to these mutagenic intermediates.
Here, we reveal that slipped junctions can assume a surprising number
of interconverting conformations where the strand opposite the slip-out
either is fully base paired or has one or two unpaired nucleotides.
These unpaired nucleotides can also arise opposite either of the nonslipped
junction arms. Junction conformation can affect binding by various
structure-specific DNA repair proteins and can also alter correct
nick-directed repair levels. Junctions that have the potential to
contain unpaired nucleotides are repaired with a significantly higher
efficiency than constrained fully paired junctions. Surprisingly,
certain junction conformations are aberrantly repaired to expansion
mutations: misdirection of repair to the non-nicked strand opposite
the slip-out leads to integration of the excess slipped-out repeats
rather than their excision. Thus, slipped-junction structure can determine
whether repair attempts lead to correction or expansion mutations