Genomic instability in cancer: molecular mechanisms and therapeutic potentials

: DNA damage usually happens in all cell types, which may originate from endogenous sources, (i.e., DNA replication errors) or be emanated from radiations or chemicals. These damages range from changes in few nucleotides to large structural abnormalities on chromosomes and, if not repaired, could disturb the cellular homeostasis or cause cell death. DNA repair, as the most significant response to DNA damage, provides biological pathways by which DNA damages are corrected and returned into their natural circumstance. However, aberration in the DNA repair mechanisms may result in genomic and chromosomal instability and the accumulation of mutations. The activation of oncogenes and/or inactivation of tumor suppressor genes are serious consequence of genomic and chromosomal instability and may bring the cells into a cancerous phenotype. Therefore, genomic and chromosomal instability is usually considered as a crucial factor in the carcinogenesis and an important hallmark of various human malignancies. In the present study, we review our current understanding of the most updated mechanisms underlying genomic instability in cancer and discuss about the potential promises of these mechanisms in finding new targets for the treatment of cancer.

PprI: The Key Protein in Response to DNA Damage in Deinococcus

Deoxyribonucleic acid (DNA) damage response (DDR) pathways are essential for maintaining the integrity of the genome when destabilized by various damaging events, such as ionizing radiation, ultraviolet light, chemical or oxidative stress, and DNA replication errors. The PprI–DdrO system is a newly identified pathway responsible for the DNA damage response in Deinococcus, in which PprI (also called IrrE) acts as a crucial component mediating the extreme resistance of these bacteria. This review describes studies about PprI sequence conservation, regulatory function, structural characteristics, biochemical activity, and hypothetical activation mechanisms as well as potential applications.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2′-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Oxidative damage diminishes mitochondrial DNA polymerase replication fidelity

Mitochondrial DNA (mtDNA) resides in a high ROS environment and suffers more mutations than its nuclear counterpart. Increasing evidence suggests that mtDNA mutations are not the results of direct oxidative damage, rather are caused, at least in part, by DNA replication errors. To understand how the mtDNA replicase, Pol γ, can give rise to elevated mutations, we studied the effect of oxidation of Pol γ on replication errors. Pol γ is a high fidelity polymerase with polymerase (pol) and proofreading exonuclease (exo) activities. We show that Pol γ exo domain is far more sensitive to oxidation than pol; under oxidative conditions, exonuclease activity therefore declines more rapidly than polymerase. The oxidized Pol γ becomes editing-deficient, displaying a 20-fold elevated mutations than the unoxidized enzyme. Mass spectrometry analysis reveals that Pol γ exo domain is a hotspot for oxidation. The oxidized exo residues increase the net negative charge around the active site that should reduce the affinity to mismatched primer/template DNA. Our results suggest that the oxidative stress induced high mutation frequency on mtDNA can be indirectly caused by oxidation of the mitochondrial replicase.

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

Anti-aging Effect of Free Curcumin and Niosome Entrapping Curcumin in H2O2-induced Aging in Human Fibroblast Cell Lines

Aging is not a disease but it a changes that come with time, aging may make an individual susceptible to disease. Exposure to smoke, UV radiation, chemicals or other exogenous agents over time can damage cells, as can produce DNA replication errors or oxidative stress. The use of antioxidant curcumin can produce intracellular defenses in addition to low expensive and no side effect. In the present study, hydrogen peroxide (H2O2) was used to produce oxidative stress in Human Fibroblast cell lines (HFB4) and free curcumin and niosome entrapping curcumin treatments are used for aged HFB4 cell lines. Using Flowcytometry analysis, p 16 INK4a and p53 BP1 genes levels were investigated as aging biomarkers. Result show that H2O2 treated cell lines are highly elevate p16 INK4a and p53 BP1 genes. While HFB4 cells treated with free curcumin and niosome entrapping curcumin were significantly protected against aged process.

Genes

Two types of nucleic acids, DNA and RNA, carry genetic information of organisms across generations. Many researchers are credited with the early work that laid the foundation of the discovery of the structure of DNA. During cell division, the cell replicates its DNA and organelles during the synthesis (S) phase of the cell cycle. Four main steps are involved in the processes of replication. DNA replication errors and cells have evolved a complex system of fixing most (but not all) of those replication errors proofreading and mismatch repair. With repeated cell division, the DNA molecule shortens with the loss of critical genes, leading to cell death. In gonads, a special enzyme called telomerase lengthens telomeres from its own RNA sequence which serves as a template to synthesize new telomeres. Although most DNA is packaged within the nucleus, mitochondria have a small amount of their own DNA called mitochondrial DNA. This chapter explores this aspect of genes.

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

Mutation Vulnerability Characterizes Human Cancer Genes

Recent studies by Tomasetti et al. revealed that the risk disparity among different types of cancer is mainly determined by inherent patterns in DNA replication errors rather than environmental factors. In this study we reveal that inherent patterns of DNA mutations plays a similar role in cancer at the molecular level. Cancer results from stochastic DNA mutations, yet non-random patterns of cancer mutations emerge when we look across hundreds of cancer genomes. Over 500 cancer genes have been identified to date as the hot spot genes of cancer mutations. It is generally believed that these gene are mutated more frequently because they reside in functionally important pathways and are hence selected during the somatic evolution process of tumor progression. This theory however does not explain why many genes in the same pathways of cancer genes are not mutated in cancer. In this study, we challenge this view by showing that the inherent patterns of spontaneous mutations of human genes not only distinguish cancer causing genes and non-cancer genes but also shapes the mutation profile of cancer genes at the sub-gene level.