Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease

Abstract

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen–modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex ∼3 bases from the 3′-side and ∼9 bases from the 5′-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

UvrA expression of Lactococcus lactis NZ9000 improve multiple stresses tolerance and fermentation of lactic acid against salt stress

“…However, the UvrA expression level of L. lactis increased slightly as heat stress continued. It seems that UvrA plays an important role in assisting the organism's adaptation to heat stresses, possibly by protecting proteins from denaturation (Christensen et al 2008). Our data shown that over expression of UvrA and exposed to heat treatment significantly enhances the growth performance and survival rate of L. lactisUvrA.…”

Site-Specific DNA Photocleavage and Photomodulation by Oligonucleotide Conjugates

“…A report by Guieysse et al (2000) confirmed this mechanism of triplex-mediated incision inhibition. However, a more recent study from the Vasquez Laboratory (Christensen et al, 2008) suggests that triplex formation does not inhibit the incision site at which the TFO is bound to and is further accessed by UvrABC nucleases. A more detailed study on a variety of DNA mutations revealed that the frequency of deletions is independent on ERCC1-XPF and TCR activity.…”

The ERCC1 and ERCC4 (XPF) genes and gene products

“…In bacteria, a psoralen ICL (Van Houten et al, 1986) and a psoralen ICL directed by a triplex-forming oligonucleotide (Christensen et al, 2008) can be repaired by dual incision via the NER pathway. It is possible that this also occurs in mammalian cells.…”