DNA damage, neurodegeneration and mitochondrial dysfunction

Due to external and internal exposures, cells accumulate enormous amounts of damage to DNA every day. This damage can be the cause of aging, neurodegeneration and cancer, or cell death, if it persists in the genome. DNA damage triggers a large cascade of events that include a variety of processes and signaling pathways including DNA repair. Cells have intricate and efficient processes for DNA repair to prevent these outcomes, but it is a matter of time. The damage accumulates both in the nuclear and mitochondrial DNA, and mitochondrial function is affected by both types. Lately, evidence supports that there is a signaling cascade from the nuclear DNA damage to mitochondrial function, and thus directly to the bioenergetic regulation of cells. This is seen across species from human to worms. We find that some DNA repair defective diseases with severe neurodegeneration have mitochondrial defects. Our studies involve cell lines, the worm (c.elegans), and mouse models and include the conditions Xeroderma pigmentosum group A, Cockaynes syndrome, Ataxia telangiectasia and others. We find a pattern of hyper parylation, deficiency in the NAD+and Sirtuin signaling, and mitochondrial stress. This involves a defect in the ability remove damaged mitochondria, a process called mitophagy, which is a sub pathway of autophagy. We are pursuing mechanistic studies of this signaling and also find that interventions at different steps can improve mitochondrial health and neurodegeneration. The mechanisms involved and some promising interventions incl

DNA-Related Pathways Defective in Human Premature Aging

One of the major issues in studies on aging is the choice of biological model system. The human premature aging disorders represent excellent model systems for the study of the normal aging process, which occurs at a much earlier stage in life in these individuals than in normals. The patients with premature aging also get the age associated diseases at an early stage in life, and thus age associated disease can be studied as well. It is thus of great interest to understand the molecular pathology of these disorders

Human premature aging, DNA repair and RecQ helicases

Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects

Metabolism, Genomics, and DNA Repair in the Mouse Aging Liver

The liver plays a pivotal role in the metabolism of nutrients, drugs, hormones, and metabolic waste products, thereby maintaining body homeostasis. The liver undergoes substantial changes in structure and function within old age. Such changes are associated with significant impairment of many hepatic metabolic and detoxification activities, with implications for systemic aging and age-related disease. It has become clear, using rodent models as biological tools, that genetic instability in the form of gross DNA rearrangements or point mutations accumulate in the liver with age. DNA lesions, such as oxidized bases or persistent breaks, increase with age and correlate well with the presence of senescent hepatocytes. The level of DNA damage and/or mutation can be affected by changes in carcinogen activation, decreased ability to repair DNA, or a combination of these factors. This paper covers some of the DNA repair pathways affecting liver homeostasis with age using rodents as model systems

Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2

The ends of linear chromosomes are capped by protein–DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2

Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops

Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere maintenance. The DNA-PK complex also functions in both DNA double strand break repair and telomere maintenance. Interaction between WRN and the DNA-PK complex has been reported in DNA double strand break repair, but their possible cooperation at telomeres has not been reported. This study analyzes thein vitro and in vivo interaction at the telomere between WRN and DNA-PKcs, the catalytic subunit of DNA-PK. The results show that DNA-PKcs selectively stimulates WRN helicase but not WRN exonuclease in vitro, affecting that WRN helicase unwinds and promotes the release of the full-length invading strand of a telomere D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo

The DNA Structure and Sequence Preferences of WRN Underlie Its Function in Telomeric Recombination Events

Telomeric abnormalities caused by loss of function of the RecQ helicase WRN are linked to the multiple premature ageing phenotypes that characterize Werner syndrome. Here we examine WRN\u27s role in telomeric maintenance, by comparing its action on a variety of DNA structures without or with telomeric sequences. Our results show that WRN clearly prefers to act on strand invasion intermediates in a manner that favours strand invasion and exchange. Moreover, WRN unwinding of these recombination structures is further enhanced when the invading strand contains at least three G-rich single-stranded telomeric repeats. These selectivities are most pronounced at NaCl concentrations within the reported intranuclear monovalent cation concentration range, and are partly conferred by WRN\u27s C-terminal region. Importantly, WRN\u27s specificity for the G-rich telomeric sequence within this precise structural context is particularly relevant to telomere metabolism and strongly suggests a physiological role in telomeric recombination processes, including T-loop dynamics

Cockayne syndrome B protein stimulates apurinic endonuclease 1 activity and protects against agents that introduce base excision repair intermediates

The Cockayne syndrome B (CSB) protein—defective in a majority of patients suffering from the rare autosomal disorder CS—is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP–DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2′-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates

Cockayne syndrome group B protein has novel strand annealing and exchange activities

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, severe neurological abnormalities and prageroid symptoms. The CS complementation group B (CSB) protein is involved in UV-induced transcription coupled repair (TCR), base excision repair and general transcription. CSB also has a DNA-dependent ATPase activity that may play a role in remodeling chromatin in vivo. This study reports the novel finding that CSB catalyzes the annealing of complementary single-stranded DNA (ssDNA) molecules with high efficiency, and has strand exchange activity. The rate of CSB-catalyzed annealing of complementary ssDNA is 25-fold faster than the rate of spontaneous ssDNA annealing under identical in vitro conditions and the reaction occurs with a high specificity in the presence of excess non-homologous ssDNA. The specificity and intrinsic nature of the reaction is also confirmed by the observation that it is stimulated by dephosphorylation of CSB, which occurs after UV-induced DNA damage, and is inhibited in the presence of ATPγS. Potential roles of CSB in cooperation with strand annealing and exchange activities for TCR and homologous recombination are discussed