Andrei Seluanov

SIRT6 Promotes DNA Repair Under Stress by Activating PARP1

A genome stability regulator integrates DNA repair and stress signaling pathways. Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)–ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress.

High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat

The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat’s cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.

DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomally-integrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G1 to G2/M (G1 < S < G2/M). HR is nearly absentin G1, most active in the S phase, and declines in G2/M. Thus, inG2/M NHEJ is elevated, while HR is on decline. This is in contrastto a general belief that NHEJ is most active in G1, while HR isactive in S, G2 and M. The overall efficiency of NHEJ was higherthan HR at all cell cycle stages. We conclude that human somaticcells utilize error-prone NHEJ as the major DSB repair pathway atall cell cycle stages, while HR is used, primarily, in the S phase.

Comparison of nonhomologous end joining and homologous recombination in human cells

The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR leads to accurate repair, while NHEJ is intrinsically mutagenic. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Here we provide a comparison of the kinetics and relative contributions of HR and NHEJ in normal human cells. We used chromosomally integrated fluorescent reporter substrates for real-time in vivo monitoring of the NHEJ and HR. By examining multiple integrated clones we show that the efficiency of NHEJ and HR is strongly influenced by chromosomal location. Furthermore, we show that NHEJ of compatible ends (NHEJ-C) and NHEJ of incompatible ends (NHEJ-I) are fast processes, which can be completed in approximately 30 min, while HR is much slower and takes 7h or longer to complete. In actively cycling cells NHEJ-C is twice as efficient as NHEJ-I, and NHEJ-I is three times more efficient than HR. Our results suggest that NHEJ is a faster and more efficient DSB repair pathway than HR.

Changes in DNA repair during aging

DNA is a precious molecule. It encodes vital information about cellular content and function. There are only two copies of each chromosome in the cell, and once the sequence is lost no replacement is possible. The irreplaceable nature of the DNA sets it apart from other cellular molecules, and makes it a critical target for age-related deterioration. To prevent DNA damage cells have evolved elaborate DNA repair machinery. Paradoxically, DNA repair can itself be subject to age-related changes and deterioration. In this review we will discuss the changes in efficiency of mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER) and double-strand break (DSB) repair systems during aging, and potential changes in DSB repair pathway usage that occur with age. Mutations in DNA repair genes and premature aging phenotypes they cause have been reviewed extensively elsewhere, therefore the focus of this review is on the comparison of DNA repair mechanisms in young versus old.

Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat

The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed “early contact inhibition.” Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27Kip1. In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16Ink4a. Furthermore, we show that the roles of p16Ink4a and p27Kip1 in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16Ink4a, and regular contact inhibition is controlled by p27Kip1. We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat.

FtsY, the Prokaryotic Signal Recognition Particle Receptor Homologue, Is Essential for Biogenesis of Membrane Proteins

In mammalian cells, many secretory proteins are targeted to the endoplasmic reticulum co-translationally, by the signal recognition particle (SRP) and its receptor. In Escherichia coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally. Unexpectedly, despite this variance, E. coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery. In this study, we investigated the possibility that the prokaryotic SRP-like machinery is involved in biogenesis of membrane proteins in E. coli. The data presented here demonstrate that the SRP-receptor homologue, FtsY, is indeed essential for expression of integral membrane proteins in E. coli, indicating that, in the case of this group of proteins, FtsY and the mammalian SRP receptor have similar functions.

Telomerase activity coevolves with body mass not lifespan.

In multicellular organisms, telomerase is required to maintain telomere length in the germline but is dispensable in the soma. Mice, for example, express telomerase in somatic and germline tissues, while humans express telomerase almost exclusively in the germline. As a result, when telomeres of human somatic cells reach a critical length the cells enter irreversible growth arrest called replicative senescence. Replicative senescence is believed to be an anticancer mechanism that limits cell proliferation. The difference between mice and humans led to the hypothesis that repression of telomerase in somatic cells has evolved as a tumor‐suppressor adaptation in large, long‐lived organisms. We tested whether regulation of telomerase activity coevolves with lifespan and body mass using comparative analysis of 15 rodent species with highly diverse lifespans and body masses. Here we show that telomerase activity does not coevolve with lifespan but instead coevolves with body mass: larger rodents repress telomerase activity in somatic cells. These results suggest that large body mass presents a greater risk of cancer than long lifespan, and large animals evolve repression of telomerase activity to mitigate that risk.

Change of the Death Pathway in Senescent Human Fibroblasts in Response to DNA Damage Is Caused by an Inability To Stabilize p53

ABSTRACT The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.

SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age

L1 retrotransposons are an abundant class of transposable elements which pose a threat to genome stability and may play a role in age-related pathologies such as cancer. Recent evidence indicates that L1s become more active in somatic tissues during the course of aging; the mechanisms underlying this phenomenon remain unknown, however. Here we report that the longevity regulating protein, SIRT6, is a powerful repressor of L1 activity. Specifically, SIRT6 binds to the 5′UTR of L1 loci, where it mono-ADP ribosylates the nuclear corepressor protein, KAP1, and facilitates KAP1 interaction with the heterochromatin factor, HP1α, thereby contributing to the packaging of L1 elements into transcriptionally repressive heterochromatin. During the course of aging, and also in response to DNA damage, however, we find that SIRT6 is depleted from L1 loci, allowing for the activation of these previously silenced retroelements.