Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.

SUMMARY The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structureactivity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

The DNA-binding box of human SPARTAN contributes to the targeting of Pol to DNA damage sites, DNA Repair http://dx.doi.org/10.1016/j. dnarep.2016.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractInappropriate repair of UV-induced DNA damage results in human diseases such as Xeroderma pigmentosum (XP), which is associated with an extremely high risk of skin cancer. A variant form of XP is caused by the absence of Polη, which is normally able to bypass UV-induced DNA lesions in an errorfree manner. However, Polη is highly error prone when replicating undamaged DNA and, thus, the regulation of the proper targeting of Polη is crucial for the prevention of mutagenesis and UV-induced cancer formation. Spartan is a novel regulator of the damage tolerance pathway, and its association with Ub-PCNA has a role in Polη targeting; however, our knowledge about its function is only rudimentary. Here, we describe a new biochemical property of purified human SPARTAN by showing that it is a DNAbinding protein. Using a DNA binding mutant, we provide in vivo evidence that DNA binding by SPARTAN regulates the targeting of Polη to damage sites after UV exposure, and this function contributes highly to its DNA-damage tolerance function.

Egy DNS-hiba következtében elakadt replikációs villa menekítése többféleképpen is megvalósulhat a sejtekben. Az, hogy melyik útvonal aktív, szigorú szabályozás alatt áll, hiszen nem mindegy, hogy a sejtekben egy gyors, ám mutagén transzléziós szintézis zajlik vagy éppen egy bonyolult struktúrákon végbemenő, de pontos másolást lehetővé tevő templátváltás. Ezen mechanizmusok közötti molekuláris kapcsoló a PCNA fehérje, illetve annak poszttranszlációs módosításai. Míg ubiquitilációja a DNS-hibatolerancia útvonalakat aktiválja, addig SUMOilációja a rekombináció-függő folyamatot gátolja. Utóbbiban élesztőben végzett vizsgálatok alapján fontos szerepet tölt be az Srs2 fehérje, több szinten hatva. Helikáz aktivitása révén részt vesz a D-hurok szétszerelésében, SUMOilált PCNA-függő módon pedig a D-hurok meghosszabbítását gátolja. Humán sejtekben ezeket a funkciókat több fehérje látja el, melyek közül a PARI-ról mutatták ki, hogy kölcsönhatásba lép a SUMOilált PCNA-vel. Munkánk során azt vizsgáltuk, hogy a PARI fehérje milyen molekuláris mechanizmusok révén valósítja meg a homológ rekombináció gátlását az elakadt replikációs villa menekítésekor: a D-hurok meghosszabbításának gátlása során megakadályozza, hogy a homológ szakaszról hosszasan történjen meg a duplikáció, lecsökkentve az esélyét az átkereszteződéseknek és meggátolva a további rekombinációs eseményeket és az esetleges genomi átrendeződéseket.

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