| Impairment of DNA replication is one of the major contributors towards genomic instability. The factors that affect the replication fork's progression can be exogenous, genetic and intrinsic. In the latter case, attenuation of DNA replication can be due to collisions with transcription, certain DNA binding proteins or unusual structures of the template DNA. This study focuses on the mechanisms of the replication inhibition caused by collisions with the transcription machinery and DNA binding proteins.; We show that head-on collisions with the elongating RNA polymerase appear to inhibit progression of the bacterial replication fork to a much greater extent than co-directional collisions. We evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries and observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude therefore, that the replication fork is attenuated upon direct physical contact with the head-on transcription machinery.; We further extend our studies on transcription regulatory elements. We create a steadfast transcription initiation complex by altering the initial transcribed sequence of the T7 A1 promoter so that it abolishes RNA polymerase's transition to the productive elongation mode in vivo. We show that this steadfast transcription initiation complex inhibits the replication fork progression in an orientation-dependent manner, i.e. during head-on collisions. Transcription terminators also appear to attenuate DNA replication, but in the opposite, co-directional orientation. Based on these data, we propose that transcription regulatory signals can serve as "punctuation marks" for DNA replication by stalling the replication fork upon its passage through the coding area in either direction.; We demonstrate that the d(GA)n/d(TC)n repeat inhibits DNA replication in E. coli in a length-dependent and orientation-independent manner. We show that this inhibition is due to the binding of multiple molecules of the F-factor encoded protein TraY that wrap DNA around themselves and form nucleosome-like structure. We believe that TraY binding to the d(GA)n/d(TC)n repeat could represent the first defined example of the "passive" replication inhibition by a protein aggregate. It also helps to understand the principles of replication inhibition by microsatellites in eukaryotic genomes. |
