Endogenously generated DNA nucleobase modifications source,and significance as possible biomarkers of malignant transformation risk,and role in anticancer therapy

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease – what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

Insights from xanthine and uracil DNA glycosylase activities of bacterial and human SMUG1: switching SMUG1 to UDG

Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only UDGs but also xanthine DNA glycosylases (XDGs). In addition, mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2 for XDG and UDG activities. Mutations at M57 (M57L) and H210 (H210G, H210M, and H210N), both of which are involved in interactions with the C2 carbonyl oxygen in uracil or xanthine, cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1, which corresponds to a position involved in base flipping. This mutation results in an activity profile resembling a human SMUG1-like enzyme as exemplified by the retention of UDG activity on mismatched base pairs and weak XDG activity. MD simulations indicate that M57L increases the flexibility of the motif 2 loop region and specifically A214, which may account for the reduced catalytic activity. G60Y completely abolishes XDG and UDG activity, which is consistent with a modeled structure in which G60Y blocks the entry of either xanthine or uracil to the base binding pocket. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive UDG as demonstrated by the uniform excision of uracil in both double-stranded and single-stranded DNA and the complete loss of XDG activity. MD simulations indicate that a combination of a reduced free volume and altered flexibility in the active-site loops may underlie the dramatic effects of the G63P mutation on the activity profile of SMUG1. This study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency.     

BRCA1: Beyond double-strand break repair

Since its discovery, the BRCA1 tumor suppressor has been shown to play a role in multiple DNA damage response pathways. Here, we will review the involvement of BRCA1 in base-excision DNA repair and highlight its clinical implications.     

Regulation of base excision repair proteins by ubiquitylation

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.