In vivo investigation of interactions between replisome components in Escherichia coli: An expanded model for the processivity switch

Background: Protein interactions within the replisome (a highly coordinated protein complex) are crucial to maintain temporal and spatial regulation for high fidelity DNA synthesis in Escherichia coli (E. coli). A key component of these interactions is the processivity switch, ensuring smooth transition of the replicative DNA polymerase III (Pol III) between Okazaki fragments on the lagging strand. Multiple interaction studies between replisome components have been performed to indicate the essential roles of Pol III (DnaE), β-clamp, DnaB helicase, DNA and the t (DnaX) subunit for this switch.Methods: Known interacting regions of both DnaE and various truncated versions of t were chosen for co-expression in E. coli. Differences in the growth pattern of cells co-expressing various truncated versions of DnaX and DnaE, on liquid and solid media were subsequently analyzed. Based on in vivo analyses to explore the interactions between these components, an expanded model for the processivity switch is presented here.Results: The analyses suggest that residues 481-643 of t are sufficient to establish a functional interaction with the DnaB helicase and DnaE during replication, while residues 461-480 of t interact with the C-terminal tail of DnaE to disengage Pol III from the β-clamp during processivity switching. We also propose that residues 430-460 of t are involved in sensing the DNA structure required for the processivity switch.Conclusion: These observations expand the current understanding of processivity switching and help dissect the regions of t utilized for binding to different replisome components such as DnaB helicase, polymerase and DNA.Keywords: Processivity Switch; Clamp Loader; DnaE; DnaX; DnaB Helicas

Molecular surveillance of HCV mono-infection and HCV-HBV co-infection in symptomatic population at Hyderabad, Pakistan

Background: Pakistan is endemic to hepatitis B and C infections. Alarming rise in hepatitis C virus (HCV) infection has been noticed in some areas of Sindh with an increasing risk for co-infection frequency in this region.Objective: To estimate the burden of HBV/HCV infection in Hyderabad Pakistan. Methods: ELISA and Nucleic acid Amplification test were performed to detect viruses. SPSS and online calculator were used for statistical analysis.Results: From a total of 108 seropositive hepatitis patients, 36.1% (n=39) were found HCV RNA-positive. Non-significant differences were observed in the frequencies of HCV infection for both genders [OR=0.735, CI (95%) 0.307-1.761, p<0.05]. The percentage of HBV DNA detection among 108 HCV-seropositive cases was 17.9% (n=19). However, HCV-HBV co-infection in HCV-RNA positive cases was determined in 48.7% (n=19) cases with non-significant difference in both genders [OR=1.51, CI (95%) = 0.38 - 5.96, p< 0.05]. Analysis suggested weakly positive correlation between HCV mono-infection and HCV-HBV co-infection and age (r =0.184, and r =0.1231), respectively.Conclusion: The study demonstrates a high prevalence of HBV co-infection among active hepatitis C patients of Hyderabad.Keywords: HCV mono-infection, HCV-HBV co-infection, molecular surveillance, Nucleic acid Amplification Test, active hepatitis C, Hyderabad, Sindh

Molecular surveillance of HCV mono-infection and HCV-HBV co-infection in symptomatic population at Hyderabad, Pakistan

Background: Pakistan is endemic to hepatitis B and C infections. Alarming rise in hepatitis C virus (HCV) infection has been noticed in some areas of Sindh with an increasing risk for co-infection frequency in this region. Objective: To estimate the burden of HBV/HCV infection in Hyderabad Pakistan. Methods: ELISA and Nucleic acid Amplification test were performed to detect viruses. SPSS and online calculator were used for statistical analysis. Results: From a total of 108 seropositive hepatitis patients, 36.1% (n=39) were found HCV RNA-positive. Non-significant differences were observed in the frequencies of HCV infection for both genders [OR=0.735, CI (95%) 0.307-1.761, p<0.05]. The percentage of HBV DNA detection among 108 HCV-seropositive cases was 17.9% (n=19). However, HCV-HBV co-infection in HCV-RNA positive cases was determined in 48.7% (n=19) cases with non-significant difference in both genders [OR=1.51, CI (95%) = 0.38 - 5.96, p< 0.05]. Analysis suggested weakly positive correlation between HCV mono-infection and HCV-HBV co-infection and age (r =0.184, and r =0.1231), respectively. Conclusion: The study demonstrates a high prevalence of HBV co-infection among active hepatitis C patients of Hyderabad

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor