Summary Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes [TC(s)], unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a component of cellular stress responses. Stalled forks are foci for genomic instability that causes genetic alterations and can give rise to cancer. Stalled forks must be protected/remodeled/repaired and replication restarted/continued in order to maintain genomic stability. We propose to continue our analyses of replication fork stalling brought about by such factors. We ask: (i) Are replisome collisions with protein-bound R-loops more prone to stall forks than collisions with unbound R- loops? which are only transient obstacles to progression. (ii) Does replication fork reversal preserve replication potential during replication-transcription conflicts? (iii) Does the accumulation of positive superhelicity between replisomes and TCs approaching each other head-on lead to replication fork stalling and/or collapse? And (iv), how do replisomes overcome collisions with RNA polymerases that are themselves stalled by DNA template damage? We have used the MIRA mechanism to begin to transit our focus on bacterial replication systems to replication with human proteins. We will investigate the DNA sequence requirements for loading of double hexamers of the MCM proteins to DNA, as well as the effect of chromatinization of the DNA substrate on the loading reaction. We ask: (i) what are the requirements for various amino acid sequence motifs in ORC1? (ii) What role is played by ORC6 (we currently do not require this protein for loading)? And (iii) what are the effects of histone modifications in the loading reaction? We are also proceeding to reconstitute the complete replication reaction with purified human replication proteins. Such a system will afford unprecedented insight into insults to replication fork progression and cellular stress responses. Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. We propose to continue our analyses of the mechanisms by which the bacterial condensin MukBEF and the cellular decatenase topoisomerase IV cooperate to promote proper chromosome compaction and segregation. We ask: (i) Does the MukBEF complex either translocate on or extrude loops of DNA? And (ii) how does replication proceed through topological domains generated by MukB and Topo IV?