Genome Integrity Discussion Group April 2021

Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is largely driven by an alternative splicing event, centered around hTERT exon-2. Skipping of exon-2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon-2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.

DNA damage checkpoint signaling elicits numerous cellular changes to cope with genotoxic stress. However, hyperactivated checkpoint signaling is detrimental to growth by inducing persistent cell-cycle arrest. Checkpoint dampening is essential to counter this effect, but its underlying mechanisms are not fully understood. The DNA repair helicase Srs2 is implicated in avoiding checkpoint hyperactivation in budding yeast via unknown mechanisms. Here we show Srs2 dampens checkpoint signaling during DNA break induction by removing a key checkpoint sensor complex RPA from chromatin. RPA has strong affinity to single strand DNA (ssDNA), a common source of genotoxic stress, and RPA-ssDNA filament is a ubiquitous trigger for checkpoint signaling. Antagonism between Srs2 and RPA is supported by numerous suppressive interactions between their mutants. Importantly, moderately reducing RPA binding to ssDNA rescues persistent checkpoint signaling, genotoxic sensitivities, and growth defects caused by lacking Srs2 and its helicase activity. Moreover, Srs2 and its helicase activity are required for curbing RPA accumulation on chromatin, and this requirement is bypassed by reducing RPA-ssDNA binding capacity. Finally, we show that Srs2 regulation of RPA is distinct from its role in recombinational repair. We propose that Srs2 has a DNA repair-independent role in checkpoint dampening via promoting RPA recycling from chromatin and this role is critical for cell survival upon genotoxic stress.

Hepatitis B virus (HBV) chronically infects 257 million patients world wide, and leads to 1 million deaths every year. The persistence of the highly stable HBV covalently closed circular DNA (cccDNA) enables HBV infection and chronicity. The formation of cccDNA is dependent on elusive host factors to repair the relaxed circular DNA (rcDNA), which is carried into the hepatocytes as part of the HBV virion. Here, we developed a novel biochemical reconstitution system to analyze cccDNA formation. We identified five human factors that are core machinery in DNA lagging strand synthesis as essential for cccDNA formation and establishment of infection. We further elucidated the detailed molecular mechanism of the repair of HBV rcDNA to cccDNA. Our findings define key components and mechanisms in HBV cccDNA formation and reveal novel targets to block HBV rcDNA repair.

SKP1 (S-phase kinase-associated protein 1) is a core subunit of the SKP1–Cullin–F-box (SCF) ubiquitin E3 ligase. We previously identified SKP1 as one of the meiotic chromatin-associated proteins in a proteomic screen (1). SKP1 localizes to synapsed chromosome axes in meiotic germ cells and specifically to the lateral elements. SKP1 is an essential gene and its global knockout leads to embryonic lethality (2). We investigated the role of SKP1 in male meiosis by an tamoxifen-inducible (Ddx4-CreERT2) deletion approach that we previously used effectively. SKP1 is required for eviction of HORMAD proteins from synapsed synaptonemal complexes. SKP1- deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, gama-H2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. Recently, we generated Skp1cKO mice using the Stra8-Cre, which is expressed in spermatogonia prior to meiotic entry. Study of this cKO mutant reveals that SKP1 is required for synapsis formation and counteracts DSB formation by keeping HORMAD proteins in check. In conclusion, SKP1 plays multiple essential roles during meiosis: synapsis initiation, synapsis maintenance, modulation of DSB formation, pachytene exit, and metaphase competence acquisition.