The linker or H1 histones are a family of nuclear proteins that is crucial to packaging DNA into nuclesosomes and for the formation of compact higher-order chromatin structures. In mammals, there are at least eleven different, evolutionarily conserved, linker histone isotypes, although the functional significance for this heterogeneity remains unclear.  It was found relatively recently that linker histones are mobile within the nucleus, interacting tightly but transiently with DNA at a particular chromatin site then unbinding moving to a new location.  Release of H1 from chromatin leads to local chromatin relaxation, an essential process for the repair of DNA in response to damage. Our hypothesis is that the minor  linker histone isotypes H1a and H1b play a role in this process.  We first constructed cell lines expressing fluorescently-tagged versions of H1a or H1b and performed FRAP (Florescent Recovery After Photobleaching) assays to determine the relative mobility of each respective linker histone isotype.  The results showed that H1b was much less mobile and therefore had a higher binding affinity for DNA than isotype H1a.  We then produced another set of cell lines that overexpressed large amounts of untagged H1a or H1b.  Into these lines we introduced fluorescently-tagged versions of PARP1 or PCNA, two proteins that are recruited to DNA in response to damage. We used micro-irradiation to monitor the kinetics of PCNA and PARP1 recruitment to localized sites of DNA damage. We observed that the overexpression of H1b, and to a lesser degree H1a, resulted in significantly slower recruitment of PARP1 and PCNA relative to control cell lines, indicative of impaired DNA damage repair. We conclude that modulation of DNA compaction by linker histone isotypes impacts the efficiency of DNA repair.

Role of linker histones in DNA damage repair

247th National Meeting (2014)

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