Yanbin Zhang

Preferential Incorporation of G Opposite Template T by the Low-Fidelity Human DNA Polymerase ι

ABSTRACT DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Polι, encoded by the RAD30B gene. We show that human Polι violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Polι preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Polι compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Polι to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Polι copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Polι incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Polι in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Polι may additionally play a specialized function in human biology.

Identification and characterization of OGG1 mutations in patients with Alzheimer's disease

Patients with Alzheimers disease (AD) exhibit higher levels of 8-oxo-guanine (8-oxoG) DNA lesions in their brain, suggesting a reduced or defective 8-oxoG repair. To test this hypothesis, this study investigated 14 AD patients and 10 age-matched controls for mutations of the major 8-oxoG removal gene OGG1. Whereas no alterations were detected in any control samples, four AD patients exhibited mutations in OGG1, two carried a common single base (C796) deletion that alters the carboxyl terminal sequence of OGG1, and the other two had nucleotide alterations leading to single amino acid substitutions. In vitro biochemical assays revealed that the protein encoded by the C796-deleted OGG1 completely lost its 8-oxoG glycosylase activity, and that the two single residue-substituted OGG1 proteins showed a significant reduction in the glycosylase activity. These results were consistent with the fact that nuclear extracts derived from a limited number of AD patients with OGG1 mutations exhibited greatly reduced 8-oxoG glycosylase activity compared with age-matched controls and AD patients without OGG1 alterations. Our findings suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD patients and provide new insight into the molecular basis for the disease.

Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase μ

ABSTRACT DNA polymerase μ (Polμ) is a newly identified member of the polymerase X family. The biological function of Polμ is not known, although it has been speculated that human Polμ may be a somatic hypermutation polymerase. To help understand the in vivo function of human Polμ, we have performed in vitro biochemical analyses of the purified polymerase. Unlike any other DNA polymerases studied thus far, human Polμ catalyzed frameshift DNA synthesis with an unprecedentedly high frequency. In the sequence contexts examined, −1 deletion occurred as the predominant DNA synthesis mechanism opposite the single-nucleotide repeat sequences AA, GG, TT, and CC in the template. Thus, the fidelity of DNA synthesis by human Polμ was largely dictated by the sequence context. Human Polμ was able to efficiently extend mismatched bases mainly by a frameshift synthesis mechanism. With the primer ends, containing up to four mismatches, examined, human Polμ effectively realigned the primer to achieve annealing with a microhomology region in the template several nucleotides downstream. As a result, human Polμ promoted microhomology search and microhomology pairing between the primer and the template strands of DNA. These results show that human Polμ is much more prone to cause frameshift mutations than base substitutions. The biochemical properties of human Polμ suggest a function in nonhomologous end joining and V(D)J recombination through its microhomology searching and pairing activities but do not support a function in somatic hypermutation.

The anti-obesity effects of green tea in human intervention and basic molecular studies.

Many researchers have reported that obesity is a major risk factor for diabetes, cardiovascular diseases, several forms of cancer (such as breast, colon and prostate), pulmonary, osteoarticular and metabolic diseases in the past decades. Recently, the hypolipidemic and anti-obesity effects of green tea in animals and humans have slowly become a hot topic in nutritional and food science research. This review will up-date the information of the anti-obesity effects of green tea in human intervention and animal studies. During recent years, an increasing number of clinical trials have confirmed the beneficial effects of green tea on obesity. However, the optimal dose has not yet been established owing to the very different results from studies with a similar design, which may be caused by differences in the extent of obesity, dietary intake, physical activity intensity, the strength of subjects’ compliance to test instruction, the genetic background of populations, body composition and dietary habits. Therefore, further investigations on a larger scale and with longer periods of observation and tighter controls are needed to define optimal doses in subjects with varying degrees of metabolic risk factors and to determine differences in beneficial effects among diverse populations. Moreover, data from laboratory studies have shown that green tea has important roles in fat metabolism by reducing food intake, interrupting lipid emulsification and absorption, suppressing adipogenesis and lipid synthesis and increasing energy expenditure via thermogenesis, fat oxidation and fecal lipid excretion. However, the exact molecular mechanisms remain elusive.

FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures

In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200–1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001–1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.

Regulation of replication protein A functions in DNA mismatch repair by phosphorylation.

Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase δ-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.

The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis

The Rev1-Polζ pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N6-ethenoadenine were severely deficient. Specifically, the predominant A→G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N6-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.

SLX4 contributes to telomere preservation and regulated processing of telomeric joint molecule intermediates

SLX4 assembles a toolkit of endonucleases SLX1, MUS81 and XPF, which is recruited to telomeres via direct interaction of SLX4 with TRF2. Telomeres present an inherent obstacle for DNA replication and repair due to their high propensity to form branched DNA intermediates. Here we provide novel insight into the mechanism and regulation of the SLX4 complex in telomere preservation. SLX4 associates with telomeres throughout the cell cycle, peaking in late S phase and under genotoxic stress. Disruption of SLX4s interaction with TRF2 or SLX1 and SLX1s nuclease activity independently causes telomere fragility, suggesting a requirement of the SLX4 complex for nucleolytic resolution of branched intermediates during telomere replication. Indeed, the SLX1–SLX4 complex processes a variety of telomeric joint molecules in vitro. The nucleolytic activity of SLX1-SLX4 is negatively regulated by telomeric DNA-binding proteins TRF1 and TRF2 and is suppressed by the RecQ helicase BLM in vitro. In vivo, in the presence of functional BLM, telomeric circle formation and telomere sister chromatid exchange, both arising out of nucleolytic processing of telomeric homologous recombination intermediates, are suppressed. We propose that the SLX4-toolkit is a telomere accessory complex that, in conjunction with other telomere maintenance proteins, ensures unhindered, but regulated telomere maintenance.

Assembling an orchestra: Fanconi anemia pathway of DNA repair.

Fanconi anemia (FA) is a recessive genetic disorder characterized by developmental defects, bone marrow failure, and cancer susceptibility. The complete set of FA genes has only been identified recently and seems to be uniquely conserved among vertebrates. Fanconi anemia proteins have been implicated in the repair of interstrand DNA crosslinks that block DNA replication and transcription. Although all thirteen FA complementation groups show similar clinical and cellular phenotypes, approximately 85% of patients presented defective FANCA, FANCC, or FANCG. The established DNA interacting components (FANCM, FANCI, FANCD2, and FANCJ) account only for approximately 5% of all FA patients, an observation that raises doubt concerning the roles of FA proteins in DNA repair. In recent years, rapid progress in the area of FA research has provided great insights into the critical roles of FA proteins in DNA repair. However, many FA proteins do not have identifiable domains to indicate how they contribute to biological processes, particularly DNA repair. Therefore, future biochemical studies are warranted to understand the biological functions of FA proteins and their implications in human diseases.

Base excision repair of oxidative DNA damage coupled with removal of a CAG repeat hairpin attenuates trinucleotide repeat expansion

Trinucleotide repeat (TNR) expansion is responsible for numerous human neurodegenerative diseases. However, the underlying mechanisms remain unclear. Recent studies have shown that DNA base excision repair (BER) can mediate TNR expansion and deletion by removing base lesions in different locations of a TNR tract, indicating that BER can promote or prevent TNR expansion in a damage location–dependent manner. In this study, we provide the first evidence that the repair of a DNA base lesion located in the loop region of a CAG repeat hairpin can remove the hairpin, attenuating repeat expansion. We found that an 8-oxoguanine located in the loop region of CAG hairpins of varying sizes was removed by OGG1 leaving an abasic site that was subsequently 5′-incised by AP endonuclease 1, introducing a single-strand breakage in the hairpin loop. This converted the hairpin into a double-flap intermediate with a 5′- and 3′-flap that was cleaved by flap endonuclease 1 and a 3′-5′ endonuclease Mus81/Eme1, resulting in complete or partial removal of the CAG hairpin. This further resulted in prevention and attenuation of repeat expansion. Our results demonstrate that TNR expansion can be prevented via BER in hairpin loops that is coupled with the removal of TNR hairpins.