I went to the Keystone Symposia ”Genomic Instability and DNA Repair” in Santa Fe, NM, USA on April 2-5, 2017 which was a joint conference with “DNA Replication and Recombination”. The focus was on basic mechanisms of DNA damage repair but also on genomic instability of cancer cells. Some of the sessions were joint session between the two conferences and it was also possible to attend presentations on either conference since the lectures halls were next to each other.
The conference started with an interesting Keynote session with Tatsuya Hirano from RIKEN Institute, Japan, presenting their system to reconstitute chromosome organization with purified mitotic chromatids from Xenopus sperm chromatin and the core histones H3/H4 and H2a/H2b. To reconstitute chromosome assembly, three histone chaperones (Nucleoplasmin, FACT and Nap1) is needed to form the histone octamer while Condensin 1 and Topoisomerase II together with ATP s needed to condense the chromatids and relax the supercoiling. Hirano also showed DNA-histones are needed for nucleosome organization while Condensin 1 is needed for higher order structures of chromatin such as chromatin loops, axis and chromosomes. Topoisomerase II is involved in all levels of chromatin organization. He also showed a system where they could assemble chromatids without nucleosomes but with Condensin I and II together with Topoisomeras II and mouse AsfI. Depletion of AsfI produce less compact chromatids with a hazy appearance.
The next speaker was Johannes Walter showed data with Xenopus egg extracts but focusing on DNA replication and repair. They could reconstitute DNA repair in S-phase using purified plasmids with different DNA damage lesions/obstacles and Xenopus egg extracts which is a very powerful system to study basic mechanisms of DNA repair. Walter described three different obstactles where the CMG helicase complex played a central role: 1) Fork convergence during replication termination 2) DNA-protein crosslinks and 3) Interstrand crosslinks (ICLs). He showed that the two replisomes can pass each other during fork convergence without slowing down and after that the ssDNA is filled in followed by ligation and dissociation of the CMG helicase complexes. For the ICL repair, he could show that pol zeta is responsible for trans-lesion synthesis (TLS) across the cross-link by template switching and that NEIL3 and FANC1-D2 are responsible for the unhooking by N-glycosyl cleavage. Furthermore, in the presence of CMG, the unhooking was mediated by NEIL3 while in the absence of CMG, FANC1-D2 mediated the unhooking.
In addition, there were two interesting posters from Walter’s lab. Joseph Loparo showed single particle imaging of the replication fork progression using lambda DNA and Xenopus extracts together with fluorescent labelled nucleotides. In FRAP experiment he could show that components of the NHEJ (non-homologues end-joining) XLF and XRCC4 interact with each other and can form filaments on DNA. In the second poster, Lin Deng used a lacO plasmid to which a lacR protein could be bound creating a DNA-protein lesion causing replication fork stalling when interphase Xenopus extract were added. Addition of Cyclin B-CDK1 caused fork collapse and double-strand breaks (DSBs). These were repaired by the Alt-EJ (alternative end joining) pathway in mitosis. The same mechanism was seen for replication stress by aphidicolin or ICLs.
On the same line, Anthony Cesare showed that lethal replication stress induced by high dose of aphidicolin caused mitotic cell death and this could partially be rescued by Reversine, a Mps1 inhibitor that removes the spindle-assembly checkpoint. In addition siRNA depletion of WAPL, which is involved in centromere cohesion, also reduce the replication stress induced mitotic cell death. In parallel, Aurora B and ATM dependent telomere deprotection seems to occur after replication stress. This is highly related to my project since we also study DNA damage during mitosis.
I had a poster presentation on Monday April 3 and there was some interest in the work although not so many people at the conference seem to work on oxidative DNA damage and repair.
Many talks and posters focused on telomere maintenance and repair and this is relevant to the MTH1 project since MTH1 and 8-oxo-dGTP has been shown to be involved in telomere replication. Michael Stone used magnetic tweezers to measure biophysical properties of the T-loop and D-loop of telomeres and TRF2 binding to the telomere. How the telomeres are protected to prevent DNA damage response was studied by Simon Boulton. He could show that removal of the RTEL1 helicase de-protects the telomeres and cause telomere loss and catastrophic cleavage by SLX1/4. Removal of the telomerase mTERT suppress this phenotype and PARP1 inhibiton or depletion also suppress telomere loss/dysfunction by preventing telomerase recruitment. In an interesting talk, Julia Cooper showed that telomeres have a function in centromere assembly during mitosis and to control the nuclear envelope breakdown during meiosis and mitosis. The protein Sad1 seems to be essential for centromere centrosome connection at the nuclear envelope and a temperature sensitive mutant of Sad1 prevent nuclear breakdown.
Another theme was the nuclear membrane which seems to be important during DNA repair. Gary Karpen showed that the repair of DSBs in the pericentromeric heterochromatin, which is difficult since it contains repetitive sequences, is divided into two parts. The early steps of homologues recombination such as ATRIP recruitment occurs rapidly while later stages such as Rad51 occurs after the DSBs are translocated to the nuclear membrane. The function of the proposed mechanism is to prevent Rad51 homologue searches in repetitive DNA.
Marco Foiani talked about a role of the DNA damage response kinases ATR, ATM and mTOR in regulating the integrity of the nuclear membrane. ATR-Chk1 depletion results in invaginations of the nuclear membrane and when subjected to mechanical stress, the nuclear membrane of the ATR depleted cells would burst and get fragmented as shown with live cell imaging and electron microscopy. This process seems to involve the Plectrin-Nesprin-2 tension sensing mechanism to connect ATR to the nuclear membrane. They also used atomic force microscopy to show that ATR and Chk1 depleted cells are too soft and cannot tolerate mechanical stress. Surprisingly, ATM depleted cells also have sensitive nuclear membranes but were stiffer compared to control cells. Single-stranded DNA seems not to be involved in contrast to the replication stress response and Nesprin-2 is phosphorylated by ATR, showing a novel role of these kinases.
The conference gave me a very nice overview of DNA repair and genomic organization and I am grateful to Radiumhemmets forskningsfonder that gave me the opportunity to attend this excellent meeting. Besides attending inspirational talks and poster sessions, I could interact and discuss with other scientist in the field and get new insights and ideas for my own research.