There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies.

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcriptional regulation, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair damage recognition proteins to triplex-directed psoralen DNA interstrand cross-links (ICLs). Thus, we hypothesized that HMGB1 modulates the repair of DNA damage in mammalian cells. We demonstrate here that mammalian cells lacking HMGB1 are hypersensitive to DNA damage induced by psoralen plus UVA irradiation (PUVA) or UVC radiation, showing less survival and increased mutagenesis. In addition, nucleotide excision repair efficiency is significantly decreased in the absence of HMGB1 as assessed by the repair and removal of UVC lesions from genomic DNA. We also explored the role of HMGB1 in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no histone acetylation upon DNA damage. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. These results reveal a role for HMGB1 in the error-free repair of DNA lesions. Its absence leads to increased mutagenesis, decreased cell survival, and altered chromatin reorganization after DNA damage. Because strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases.HMGB1 ͉ nucleotide excision repair ͉ ultraviolet radiation ͉ psoralen ͉ DNA interstand crosslinks H MGB1 is a highly abundant, multifunctional protein that influences chromatin structure and remodeling by binding to the internucleosomal linker regions in chromatin (1), and facilitating nucleosome sliding (2). In addition, HMGB1 has been shown to be involved in V(D)J recombination (3), transcriptional regulation (4), and in inflammation (5). Because of its role in inflammation, HMGB1 is currently being targeted for treatment of rheumatoid arthritis (6) and sepsis (7), and is also being considered as a player in the invasive and metastatic properties of cancer (8).Among its diverse functions, HMGB1 is also capable of binding to DNA damaged by carcinogens [e.g., acetylaminofluorene (9), benzo[a]pyrene diol epoxide (9) and UV light (UVC) (10)], and chemotherapeutic agents [e.g., cisplatin (11) and psoralen plus UVA (PUVA) (12)]. Binding of HMGB1 to the damaged site induces a near ninety degree bend in the DNA (11), however, the biological consequence of this interaction is not clear. If by binding to DNA lesions HMGB1 could facilitate DNA repair, the response to DNA damage, or damage-induced chromatin remodeling, then it might prevent a mutagenic and/or carcinogenic outcome of exposure to DNA damaging agents.The roles of HMGB1 in the response to two...

The high mobility group protein B1 (HMGB1) is a highly abundant protein with roles in several cellular processes, including chromatin structure and transcriptional regulation, as well as an extracellular role in inflammation. HMGB1's most thoroughly defined function is as a protein capable of binding specifically to distorted and damaged DNA, and its ability to induce further bending in the DNA once it is bound. This characteristic in part mediates its function in chromatin structure (binding to the linker region of nucleosomal DNA and increasing the instability of the nucleosome structure) as well as transcription (bending promoter DNA to enhance the interaction of transcription factors), but the functional consequences of HMGB1's binding to damaged DNA is still an area of active investigation. In this review we describe HMGB1's actions in the nucleotide excision repair (NER) pathway, and we discuss aspects of both the "repair shielding" and "repair enhancing" hypotheses that have been suggested. We also report information regarding HMGB1's roles in the mismatch repair (MMR), non-homologous end-joining (NHEJ), and V(D)J recombination pathways, as well as its newly-discovered involvement in the base excision repair (BER) pathway. We further explore the potential of HMGB1 in DNA repair in the context of chromatin. The elucidation of HMGB1's role in DNA repair is critical for the complete understanding of HMGB1's intracellular functions, which is particularly relevant in the context of anti-HMGB1 therapies that are being developed to treat inflammatory diseases. The HMGB1 ProteinThe high mobility group B1 (HMGB1) protein (previously known as HMG1, or amphoterin), is a member of the high mobility group family of proteins. This family is separated into three groups: the HMGA (formerly HMG-I/Y) proteins, so named because they contain an A-T hook domain that binds selectively to the minor groove of AT-rich DNA; the HMGB proteins, which contain a DNA-binding B box domain that binds distorted or non-B DNA structures with high affinity and induces severe bends in the DNA; and HMGN proteins (previously named HMG-14/17), which contain a nucleosome binding domain responsible for binding to nucleosomes [1]. All of these proteins are so-called "architectural transcription factors" because they act by binding the DNA in a structuredependent manner, and modify transcriptional regulation and chromatin structure [2]. A number of comprehensive reviews have been written about the activity of the HMG family of proteins [3,4].As an architectural nuclear factor, HMGB1 is capable of binding to the linker region of nucleosomal DNA [5,6] and it competes with histone H1 to modify the dynamics of chromatin structure [7]. In addition, HMGB1 acts as a transcriptional cofactor, enhancing . In 1999, a co-crystal structure was derived from the HMGB1 A box binding to a cisplatin-modified oligonucleotide [48], and this showed that HMGB1's binding to the damaged DNA induced a severe bend, over and above the distortion induced by the adduct...

After birth the proliferation of cardiac cells declines, and further growth of the heart occurs by hypertrophic cell growth. In the present study the cell proliferation capacity of mouse embryonic stem (ES) cells versus neonatal cardiomyocytes and the effects of reactive oxygen species (ROS) on cardiomyogenesis and cardiac cell proliferation of ES cells was investigated. Low levels of hydrogen peroxide stimulated cardiomyogenesis of ES cells and induced proliferation of cardiomyocytes derived from ES cells and neonatal mice, as investigated by nuclear translocation of cyclin D1, downregulation of p27Kip1, phosphorylation of retinoblastoma (Rb), increase of Ki-67 expression and incorporation of BrdU. The observed effects were blunted by the free radical scavengers vitamin E and 2-mercaptoglycin (NMPG). In ES cells ROS induced expression of the cardiac-specific genes encoding α-actin, β-MHC, MLC2a, MLC2v and ANP as well as the transcription factors GATA-4, Nkx-2.5, MEF2C, DTEF-1 and the growth factor BMP-10. During differentiation ES cells expressed the NADPH oxidase isoforms Nox-1, Nox-2 and Nox-4. Treatment of cardiac cells with ROS increased Nox-1, Nox-4, p22-phox, p47-phox and p67-phox proteins as well as Nox-1 and Nox-4 mRNA, indicating feed-forward regulation of ROS generation. Inhibition of NADPH oxidase with diphenylen iodonium chloride (DPI) and apocynin abolished ROS-induced cardiomyogenesis of ES cells. Our data suggest that proliferation of neonatal and ES-cell-derived cardiac cells involves ROS-mediated signalling cascades and point towards an involvement of NADPH oxidase in cardiovascular differentiation of ES cells.

Unique among translesion synthesis (TLS) DNA polymerases, pol ζ is essential during embryogenesis. To determine whether pol ζ is necessary for proliferation of normal cells, primary mouse fibroblasts were established in which Rev3L could be conditionally inactivated by Cre recombinase. Cells were grown in 2% O2 to prevent oxidative stress-induced senescence. Cells rapidly became senescent or apoptotic and ceased growth within 3–4 population doublings. Within one population doubling following Rev3L deletion, DNA double-strand breaks and chromatid aberrations were found in 30–50% of cells. These breaks were replication dependent, and found in G1 and G2 phase cells. Double-strand breaks were reduced when cells were treated with the reactive oxygen species scavenger N-acetyl-cysteine, but this did not rescue the cell proliferation defect, indicating that several classes of endogenously formed DNA lesions require Rev3L for tolerance or repair. T-antigen immortalization of cells allowed cell growth. In summary, even in the absence of external challenges to DNA, pol ζ is essential for preventing replication-dependent DNA breaks in every division of normal mammalian cells. Loss of pol ζ in slowly proliferating mouse cells in vivo may allow accumulation of chromosomal aberrations that could lead to tumorigenesis. Pol ζ is unique amongst TLS polymerases for its essential role in cell proliferation.