Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I.

The mechanism by which DNA polymerases achieve their extraordinary accuracy has been intensely studied because of the linkage between this process and mutagenesis and carcinogenesis. Here, we have used single-molecule fluorescence microscopy to study the process of nucleotide selection and exonuclease action. Our results show that the binding of Escherichia coli DNA polymerase I (Klenow fragment) to a primer-template is stabilized by the presence of the next correct dNTP, even in the presence of a large excess of the other dNTPs and rNTPs. These results are consistent with a model where nucleotide selection occurs in the open complex prior to the formation of a closed ternary complex. Our assay can also distinguish between primer binding to the polymerase or exonuclease domain and, contrary to ensemble-averaged studies, we find that stable exonuclease binding only occurs with a mismatched primer terminus

Carcinogenic adducts induce distinct DNA polymerase binding orientations

DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

We used tandem mass spectrometry to identify E. histolytica cyst proteins in 5 cyst positive stool samples. We report the identification of 417 non-redundant E. histolytica proteins including 195 proteins that were not identified in existing trophozoite derived proteome or EST datasets, consistent with cyst specificity. Because the cysts were derived directly from patient samples with incomplete purification, a limited number of proteins were identified (N = 417) that probably represent only a partial proteome. Nevertheless, the study succeeded in identifying proteins that are likely to be abundant in the cyst stage of the parasite. Several of these proteins may play roles in E. histolytica stage conversion or cyst function. Proteins identified in this study may be useful markers for diagnostic detection of E. histolytica cysts. Overall, the data generated in this study promises to aid the understanding of the cyst stage of the parasite which is vital for disease transmission and pathogenesis in E. histolytica

Intra- and Inter-Individual Differences in Adolescent Depressive Mood: the Role of Relationships with Parents and Friends

Abstract: Utilizing four waves of data from 1126 secondary school Dutch adolescents (Mage = 13.95 at the first wave; 53% boys), the current study examined the interplay between parent-adolescent and friend-adolescent relationship quality (satisfaction and conflict) in relation to adolescents’ depressive mood. Using multilevel analyses, the interacting effects of parent/friend relationship quality on depressive mood were tested at both the intra- and inter-individual level. Analyses at the intra-individual level investigated whether individual depressive mood fluctuated along with changes in their social relationships regardless of one’s general level of depressive mood; and analyses at the inter-individual level examined whether the average differences in depressive mood between adolescents were associated with different qualities of social relationships. We interpreted the patterns of interactions between parent and friend relationships using four theoretical models: the reinforcement, toxic friends, compensation, and additive model. The results demonstrate the covariation of parent- and friend- relationship quality with adolescents’ depressive mood, and highlight that parent and peer effects are not independent from each other—affirming the compensation and additive models at the intra-individual and the reinforcement and additive models at the inter-individual level. The findings highlight the robustness of the protective effects of parent and peer support and the deleterious effects of conflictual relationships for adolescent mental health. The results have implications for both the theoretical and practical design of (preventive) interventions aimed at decreasing adolescents’ depressive mood

Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment

Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism