Studies of cohesin functions in the yeast and human DNA damage response

Maintaining genome stability is critical to cell survival and normal cell growth and most human cancers display some form of genome instability. Genome instability is caused by multiple reasons and the ability to properly recognize, signal and subsequently repair DNA damages is crucial. DNA double-strand breaks (DSBs) are considered as the most toxic type of DNA lesion and therefore the ability to accurately repair these breaks are of outmost importance. The cohesin complex is a large DNA-binding complex with numerous functions vital for maintaining genome integrity. The canonical role of the cohesin complex is to mediate cohesion between the sister chromatids from the time they are generated to their separation in mitosis. However, cohesin has over the years been assigned with additional functions independent on cohesion, such as regulation of gene transcription, DSB repair and activation of DNA damage checkpoints. We have investigated the role of cohesin and its loading partner NIPBL, in the cellular responses to DSBs, using human cell cultures and budding yeast as model systems. There are two main mechanisms used to repair DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). More recently, a new “alternative” pathway for DSB repair has emerged, termed alternative end joining (AEJ). By studying B-cells derived from patients with Cornelia de Lange Syndrome, we have observed a strong correlation between heterozygous loss-of-function mutations in the NIPBL gene and a shift towards the use of the microhomology-based A-EJ mechanism for DSB repair during class switch recombination. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Our results suggest that NIPBL plays an important role for NHEJ, potentially by regulating DNA end resection. In budding yeast postreplicative cells, the cohesion is reactivated in response to a DSB. This reactivation includes additional Scc2-dependent loading of cohesin to the region around the DSB and formation of new cohesion, both proximal to the DSB and on undamaged areas of the genome. This phenomenon is known as damage-induced (DI) cohesion. By analyzing the role of DNA polymerase η in DI cohesion we discovered that establishment of DI cohesion at the vicinity of a DSB and on undamaged chromosomes, genome wide, are regulated differently. We concluded this based on our finding that Polη is required for genome-wide DI cohesion while it is dispensable for S phase cohesion and DSB-proximal cohesion. Cohesion establishment, both during S phase and following a DSB, depend on the acetylation activity of the highly conserved acetyltransferase Eco1. Using in vitro studies, we found that Polη is an Eco1 substrate. In addition, we provide results suggesting that Eco1 acetylation of Polη regulates its activity in DI-cohesion. All together, these studies highlight the importance of cohesin, and its regulators, for genome stability. Future investigations, aimed at addressing the different mechanisms by which cohesin functions in the DNA damage response will most likely advance our understanding of how genome stability is maintained. LIST OF PUBLICATIONS This thesis is based on the following articles and manuscripts. They will be referred to in the text by their roman numerals. I. Enervald, E. *, Du, L. *, Visnes, T. , Björkman, A. , Lindgren, E. , Wincent, J, Borck, G. , Colleaux, L. , Cormier-Daire, V. , van Gent, D. , Pie, J., Puisac, B. , de Miranda, N. , Kracker, S. , Hammarström, L, Villartay J.P., Durandy, A. , Jacqueline Schoumans, J., Ström, L. and Pan-Hammarström, Q. A regulatory role for the cohesin loader NIPBL in non homologous end joining during immunoglobulin class switch recombination. Accepted for publication in J. Exp. Med. 2013 * Equal contribution II. Enervald, E. *, Lindgren, E. *. , Katou, Y. , Shirahige, K. and Ström, L. Importance of Polη for damage-induced cohesion reveals differential regulation of cohesion establishment at the break site and genome-wide. PLoS Genet. 2013;9(1) * Equal contribution III. Enervald, E, Palmgren, C. , Rutishauser, D. and Ström, L. Acetylation of DNA Polymerase η by Eco1 regulates its function in genomewide damage-induced cohesion. Manuscript