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Biomedical subjects

Karen M Vasquez

Publications and source records attributed to Karen M Vasquez.

15 recordsLinked to original sources
PubMed2007

High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.

Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted.

Animals↗
PubMed2006

Targeting oncogenes to improve breast cancer chemotherapy.

Despite recent advances in treatment, breast cancer remains a serious health threat for women. Traditional chemotherapies are limited by a lack of specificity for tumor cells and the cell cycle dependence of many chemotherapeutic agents. Here we report a novel strategy to help overcome these limitations. Using triplex-forming oligonucleotides (TFOs) to direct DNA damage site-specifically to oncogenes overexpressed in human breast cancer cells, we show that the effectiveness of the anticancer nucleoside analogue gemcitabine can be improved significantly. TFOs targeted to the promoter region of c-myc directly inhibited gene expression by approximately 40%. When used in combination, specific TFOs increased the incorporation of gemcitabine at the targeted site approximately 4-fold, presumably due to induction of replication-independent DNA synthesis. Cells treated with TFOs and gemcitabine in combination showed a reduction in both cell survival and capacity for anchorage-independent growth (approximately 19% of untreated cells). This combination affected the tumorigenic potential of these cancer cells to a significantly greater extent than either treatment alone. This novel strategy may be used to increase the range of effectiveness of antitumor nucleosides in any tumor which overexpresses a targetable oncogene. Multifaceted chemotherapeutic approaches such as this, coupled with triplex-directed gene targeting, may lead to more than incremental improvements in nonsurgical treatment of breast tumors.

Adenocarcinoma↗
PubMed2006

Non-B DNA structure-induced genetic instability.

Repetitive DNA sequences are abundant in eukaryotic genomes, and many of these sequences have the potential to adopt non-B DNA conformations. Genes harboring non-B DNA structure-forming sequences increase the risk of genetic instability and thus are associated with human diseases. In this review, we discuss putative mechanisms responsible for genetic instability events occurring at these non-B DNA structures, with a focus on hairpins, left-handed Z-DNA, and intramolecular triplexes or H-DNA. Slippage and misalignment are the most common events leading to DNA structure-induced mutagenesis. However, a number of other mechanisms of genetic instability have been proposed based on the finding that these structures not only induce expansions and deletions, but can also induce DNA strand breaks and rearrangements. The available data implicate a variety of proteins, such as mismatch repair proteins, nucleotide excision repair proteins, topoisomerases, and structure specific-nucleases in the processing of these mutagenic DNA structures. The potential mechanisms of genetic instability induced by these structures and their contribution to human diseases are discussed.

Base Sequence↗
PubMed2006

Z-DNA-forming sequences generate large-scale deletions in mammalian cells.

Spontaneous chromosomal breakages frequently occur at genomic hot spots in the absence of DNA damage and can result in translocation-related human disease. Chromosomal breakpoints are often mapped near purine-pyrimidine Z-DNA-forming sequences in human tumors. However, it is not known whether Z-DNA plays a role in the generation of these chromosomal breakages. Here, we show that Z-DNA-forming sequences induce high levels of genetic instability in both bacterial and mammalian cells. In mammalian cells, the Z-DNA-forming sequences induce double-strand breaks nearby, resulting in large-scale deletions in 95% of the mutants. These Z-DNA-induced double-strand breaks in mammalian cells are not confined to a specific sequence but rather are dispersed over a 400-bp region, consistent with chromosomal breakpoints in human diseases. This observation is in contrast to the mutations generated in Escherichia coli that are predominantly small deletions within the repeats. We found that the frequency of small deletions is increased by replication in mammalian cell extracts. Surprisingly, the large-scale deletions generated in mammalian cells are, at least in part, replication-independent and are likely initiated by repair processing cleavages surrounding the Z-DNA-forming sequence. These results reveal that mammalian cells process Z-DNA-forming sequences in a strikingly different fashion from that used by bacteria. Our data suggest that Z-DNA-forming sequences may be causative factors for gene translocations found in leukemias and lymphomas and that certain cellular conditions such as active transcription may increase the risk of Z-DNA-related genetic instability.

Animals↗
PubMed2006

A web-based search engine for triplex-forming oligonucleotide target sequences.

Triplex technology offers a useful approach for site-specific modification of gene structure and function both in vitro and in vivo. Triplex-forming oligonucleotides (TFOs) bind to their target sites in duplex DNA, thereby forming triple-helical DNA structures via Hoogsteen hydrogen bonding. TFO binding has been demonstrated to site-specifically inhibit gene expression, enhance homologous recombination, induce mutation, inhibit protein binding, and direct DNA damage, thus providing a tool for gene-specific manipulation of DNA. We have developed a flexible web-based search engine to find and annotate TFO target sequences within the human and mouse genomes. Descriptive information about each site, including sequence context and gene region (intron, exon, or promoter), is provided. The engine assists the user in finding highly specific TFO target sequences by eliminating or flagging known repeat sequences and flagging overlapping genes. A convenient way to check for the uniqueness of a potential TFO binding site is provided via NCBI BLAST. The search engine may be accessed at spi.mdanderson.org/tfo.

Animals↗
PubMed2005

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.

Cross-Linking Reagents↗
PubMed2005

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.

Base Sequence↗
PubMed2005

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells.

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.

Base Pair Mismatch↗
PubMed2005

Repair of genome destabilizing lesions.

Living organisms are constantly exposed to detrimental agents both from the environment (e.g. ionizing radiation, ultraviolet light, natural and synthetic chemicals) and from endogenous metabolic processes (e.g. oxidative and hydrolytic reactions), resulting in modifications of proteins, lipids and DNA. Proteins and lipids are degraded and resynthesized, but the DNA is replicated only during cell division, when DNA damage may result in mutation fixation. Thus the DNA damage generated has the potential to lead to carcinogenesis, cell death, or other genetic disorders in the absence of efficient error-free repair. Because modifications in DNA sequence or structure may be incompatible with its essential role in preservation and transmission of genetic information from generation to generation, exquisitely sensitive DNA repair pathways have evolved to maintain genomic stability and cell viability. This review focuses on the repair and processing of genome destabilizing lesions and helical distortions that differ significantly from the canonical B-form DNA in mammalian cells. In particular, we discuss the introduction and processing of site-specific lesions in mammalian cells with an emphasis on psoralen interstrand crosslinks.

Animals↗
PubMed2004

Naturally occurring H-DNA-forming sequences are mutagenic in mammalian cells.

Naturally occurring DNA sequences can form noncanonical structures such as H-DNA, which are abundant and regulate the expression of several disease-linked genes. Here, we show that H-DNA-forming sequences are intrinsically mutagenic in mammalian cells. This finding suggests that DNA is a causative factor in mutagenesis and not just the end product. By using the endogenous H-DNA-forming sequence found in the human c-myc promoter, mutation frequencies in a reporter gene were increased approximately 20-fold over background in COS-7 cells. H-DNA-induced double-strand breaks (DSBs) were detected near the H-DNA locus. The structures of the mutants revealed microhomologies at the breakpoints, consistent with a nonhomologous end-joining repair of the DSBs. These results implicate H-DNA-induced DSBs in c-myc gene translocations in diseases such as Burkitt's lymphoma and t(12;15) BALB/c plasmacytomas, where most breakpoints are found near the H-DNA-forming site. Thus, our findings suggest that H-DNA is a source of genetic instability resulting from DSBs and demonstrate that naturally occurring DNA sequences are mutagenic in mammals, perhaps contributing to genetic evolution and disease.

Animals↗
PubMed2004

Mutation frequencies in murine keratinocytes as a function of carcinogenic status.

A link between genetic abnormalities and carcinogenesis is well established. It follows that a correlation exists between mutation frequency and malignant progression. We have determined the spontaneous and DNA damage-induced mutation frequencies for a series of cell lines derived from SENCAR mouse keratinocytes at various stages of malignant progression. Nontumorigenic mouse keratinocytes (3PC), papillomas (MT1/2), squamous-cell carcinomas (CH72), and spindle-cell carcinomas (CH72T4) were transfected with damaged or undamaged shuttle vectors containing a supF mutation reporter gene. The plasmid mutation frequencies were determined by blue/white screening. The spontaneous plasmid mutation frequency of the squamous-cell carcinoma line was slightly higher than the mutation frequencies of the other cell lines tested. The DNA damage induced by triplex-directed psoralen crosslinks increased the mutation frequencies sixfold to eighteenfold in all cell lines tested, with no significant differences among the cell lines. Sequence analyses revealed that the spindle-cell carcinoma line had a different spontaneous mutation spectrum from the other cell lines. DNA damage-induced mutations were predominantly point mutations at the triplex-duplex junction in all of the cell lines tested, as expected. These data suggested that a strong mutator phenotype was not required for progression to an advanced malignant phenotype in our model system.

Animals↗
PubMed2003

Critical DNA damage recognition functions of XPC-hHR23B and XPA-RPA in nucleotide excision repair.

It has been reported that 80-90% of human cancers may result, in part, from DNA damage. Cell survival depends critically on the stability of our DNA and exquisitely sensitive DNA repair mechanisms have developed as a result. In humans, nucleotide excision repair (NER) protects the DNA against the mutagenic effects of carcinogens and ultraviolet (UV) radiation from sun exposure. By preventing mutations from forming in the DNA, the repair machinery ultimately protects us from developing cancers. DNA damage recognition is the rate-limiting step in repair, and although many details of NER have been elucidated, the mechanisms by which DNA damage is recognized remain to be fully determined. Two primary protein complexes have been proposed as the damaged DNA recognition factor in NER: xeroderma pigmentosum protein A-replication protein A (XPA-RPA) and xeroderma pigmentosum protein C-human homolog of RAD23B (XPC-hHR23B). Here we compare the evidence that supports damage detection by these protein complexes and propose a model for DNA damage recognition in NER based on the current understanding of the roles these proteins may play in the processing of DNA lesions.

DNA↗
PubMed2002

Site-directed recombination via bifunctional PNA-DNA conjugates.

Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to single-stranded DNA via Watson-Crick base pairing and can form triple helices via Hoogsteen binding to DNAPNA duplexes. Dimeric bis-PNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote site-directed recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA-DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background. Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent linkage. In addition, the bis-PNA and the bis-PNA-DNA conjugate were found to induce DNA repair specifically in the target plasmid. Both the PNA-induced recombination and the PNA-induced repair were found to be dependent on the nucleotide excision repair factor, XPA (xeroderma pigmentosum complementation group A protein). These results suggest that the formation of a PNA clamp on duplex DNA creates a helical distortion that strongly provokes DNA repair and thereby sensitizes the target site to recombination. The ability to promote recombination in a site-directed manner using PNA-DNA conjugates may provide a useful strategy to achieve targeted correction of defective genes.

DNA↗
PubMed2002

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions.

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

Base Sequence↗