Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences.

Histone-like proteins (such as HU, H-NS, and Fis) participate in nucleoid organization and in DNA replication, recombination, and transcription. Cold shock and anoxia upregulates a homologue of HU (Hlp) in Mycobacterium smegmatis, the nonpathogenic model of Mycobacterium tuberculosis. We show using electrophoretic mobility shift assays that Hlp, which in addition to the HU fold has a basic C-terminal tail containing multiple PAKK and PAAK repeats, has very high affinity for DNA. The affinity of Hlp for 76 bp linear DNA is higher, K d = 0.037 +/- 0.001 nM, compared to an Hlp variant without the C-terminal repeats, K d = 2.5 +/- 0.1 nM and the isolated C-terminal repeat domain, K d = 0.8 +/- 0.2 nM, where K d in all cases reflects an aggregate affinity for the DNA probes, not the affinity for binding to a single site. Hlp lacking the entire C-terminal domain binds DNA only poorly. These data indicate that both Hlp domains contribute to high-affinity DNA binding. Hlp promotes DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At <100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when present together. Notably, while DNA end-joining can be achieved by the isolated C-terminal domain, transcriptional repression requires for both domains to be present on a single polypeptide. Given the low cellular concentration of Hlp, our data suggest that its primary functional role may be in DNA-dependent responses to environmental stress rather than in nucleoid organization.

In mesophilic prokaryotes, the DNA-binding protein HU participates in nucleoid organization as well as in regulation of DNA-dependent processes. Little is known about nucleoid organization in thermophilic eubacteria. We show here that HU from the hyperthermophilic eubacterium Thermotoga maritima HU bends DNA and constrains negative DNA supercoils in the presence of topoisomerase I. However, while binding to a single site occludes ∼35 bp, association of T. maritima HU with DNA of sufficient length to accommodate multiple protomers results in an apparent shorter occluded site size. Such complexes consist of ordered arrays of protomers, as revealed by the periodicity of DNase I cleavage. Association of TmHU with plasmid DNA yields a complex that is remarkably resistant to DNase I-mediated degradation. TmHU is the only member of this protein family capable of occluding a 35 bp nonspecific site in duplex DNA; we propose that this property allows TmHU to form exceedingly stable associations in which DNA flanking the kinks is sandwiched between adjacent proteins. We suggest that T. maritima HU serves an architectural function when associating with a single 35 bp site, but generates a very stable and compact aggregate at higher protein concentrations that organizes and protects the genomic DNA.

Alternative DNA structure-forming sequences can stimulate mutagenesis and are enriched at mutation hotspots in human cancer genomes, implicating them in disease etiology. However, the mechanisms involved are not well characterized. Here, we discover that Z-DNA is mutagenic in yeast as well as human cells, and that the nucleotide excision repair complex, Rad10-Rad1(ERCC1-XPF), and the mismatch repair complex, Msh2-Msh3, are required for Z-DNA-induced genetic instability in yeast and human cells. Both ERCC1-XPF and MSH2-MSH3 bind to Z-DNA-forming sequences, though ERCC1-XPF recruitment to Z-DNA is dependent on MSH2-MSH3. Moreover, ERCC1-XPF − dependent DNA strand-breaks occur near the Z-DNA-forming region in human cell extracts, and we model these interactions at the submolecular level. We propose a relationship in which these complexes recognize and process Z-DNA in eukaryotes, representing a mechanism of Z-DNA-induced genomic instability.

Many effective agents used in cancer chemotherapy cause DNA interstrand crosslinks (ICLs), which covalently link both strands of the double helix together resulting in cytotoxicity. ICLs are thought to be processed by proteins from a variety of DNA repair pathways; however, a clear understanding of ICL recognition and repair processing in human cells is lacking. Previously, we found that the high mobility group box 1 (HMGB1) protein bound to triplex-directed psoralen ICLs (TFO-ICLs) in vitro, cooperatively with NER damage recognition proteins, promoted removal of UVC-induced lesions and facilitated error-free repair of TFO-ICLs in mouse fibroblasts. Here, we demonstrate that HMGB1 recognizes TFO-ICLs in human cells, and its depletion increases ICL-induced mutagenesis in human cells without altering the mutation spectra. In contrast, HMGB1 depletion in XPA-deficient human cells significantly altered the ICL-induced mutation spectrum from predominantly T→A to T→G transversions. Moreover, the recruitment of XPA and HMGB1 to the ICLs is co-dependent. Finally, we show that HMGB1 specifically introduces negative supercoils in ICL-containing plasmids in HeLa cell extracts. Taken together, our data suggest that in human cells, HMGB1 functions in association with XPA on ICLs and facilitates the formation of a favorable architectural environment for ICL repair processing.