The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser384 by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated–induced phosphorylation at Ser384 constitutes a negative feedback loop that regulates DSB repair.

The Phillies continued to bolster their infield depth on Friday by signing versatile veteran Sean Rodriguez to a Minor League contract with an invite to big league Spring Training, MLB.com has learned.

The Phillies acquired All-Star catcher J.T. Realmuto from the Marlins on Thursday for catcher Jorge Alfaro, right-hander Sixto Sanchez, left-hander Will Stewart and $250,000 in international bonus slot money, the latest move in a busy and ambitious offseason for a team eager to return to the postseason.

Phillies pitchers and catchers have their first workout Wednesday morning at Carpenter Complex. Almost anything can happen between Wednesday and Opening Day. That said, here is a very early prediction of the Phillies' Opening Day roster, knowing the Phillies remain in the hunt to sign Bryce Harper or Manny Machado.

The Phillies introduced J.T. Realmuto, their new All-Star catcher, in a news conference Tuesday at the team's Spring Training complex in Clearwater, Fla.

Gabe Kapler is trying to keep the focus on his players in 2019, but it might be an impossible task.

A slimmed down Maikel Franco and a bulked up Scott Kingery will compete for playing time at third base this season.

Jake Arrieta on Thursday offered an unexpected explanation for his second-half struggles in 2018: he pitched much of last season with an injured left knee.

Odubel Herrera smiled and patted his stomach. The paunch that he carried into Spring Training 2018 is no longer there. Herrera has reported to camp leaner and, as he said Friday morning at Spectrum Field, motivated to bounce back from the worst season of his four-year career.

Phillies president Andy MacPhail opened a 28-minute press conference on Friday afternoon at Spectrum Field with facts and figures about investments the organization made the past few seasons as the team wallowed at the bottom of the National League. Then MacPhail talked a lot about Bryce Harper and Manny Machado as well as manager Gabe Kapler.

On this opening week of Spring Training, all 30 Major League teams have one thing in common: optimism. Here's an optimism cheat sheet for each of them.

Maikel Franco followed the endless and often mind-numbing speculation the past few months from the Dominican Republic. Franco knows that Manny Machado could show up at Spectrum Field at any moment. The Phillies entertained him at Citizens Bank Park just before Christmas. They have made him at least one contract offer. If the Phillies sign him, Franco also knows he could be traded.

Phillies manager Gabe Kapler has a little advice for the players who could be impacted most if the team signs Bryce Harper or Manny Machado.

Cesar Hernandez scored 91 runs last season, the Phillies' highest mark since Rollins scored 102 in 2012. Now the luster is no longer there for players who score 100 runs or post 100 RBIs, but Hernandez should score at least 100 runs this season, if he stays healthy and if the Phillies' offense improves as expected.

The Phillies list isn't quite as strong as it once was because of the Realmuto deal, but there's still some very good talent at the top and some exciting players on the rise.

Spring Training is imminent, Opening Day is within sight and the big league season isn't complete for fans without a subscription to MLB.TV. The most comprehensive streaming service in professional sports is now available for the 2019 season.

MLB.com looked at each team's two highest-ranked position players in WAR, according to the Steamer projections. Here are the top 10, but keep in mind that things could change once Manny Machado and Bryce Harper find homes.

It's almost time for Spring Training yet again, as Angels pitchers and catchers report to their Spring Training complex in Tempe, Ariz. on Feb. 12.

Our weekly series previewing each of baseball's six divisions begins with the American League West. Let's take a team-by-team look at the biggest questions this season.

Since debuting in 2011, center fielder Mike Trout has been far and away the best player in the Majors, but the Angels have reached the postseason just once in his eight-year career.

Brad Ausmus held his first media session of the spring on the day pitchers and catchers officially reported on Tuesday, giving updates on rehabbing players such as Shohei Ohtani, Albert Pujols and Zack Cozart.

The Angels head to Spring Training with most of their roster set, but there will be competition for a few spots. Here's a look at the projected roster for the Angels, as they begin their first campaign under manager Brad Ausmus.

Shohei Ohtani, who is aiming for a return in May as a designated hitter but won't pitch in 2019, isn't able to participate in the on-field workouts with his teammates, but has progressed to swinging the bat and working out indoors.

The 2019 MLB season feels so close now. Spring Training has begun. Players are taking the field. So it's time to rank the best of the best.

On this opening week of Spring Training, all 30 Major League teams have one thing in common: optimism. Here's an optimism cheat sheet for each of them.

Angels first baseman Albert Pujols met with the media for the first time this spring on Sunday and said he's fully healthy after undergoing arthroscopic surgery on his left knee in late August.

The Angels believe they can help newly acquired catcher Jonathan Lucroy improve both offensively and defensively.

VOLUME 279 (2004) PAGES 33438–33446For Fig. 1B, the second, third, and fifth panels were mistakenly duplicated during article preparation as no yeast colonies were observed in these conditions. The corrected images are presented in the revised Fig. 1B. This correction does not affect the results or conclusions of the work. The authors apologize for the error.jbc;295/50/17382/F1F1F1Figure 1B.

VOLUME 289 (2014) PAGES 6604–6618In Fig. 4G, in the foxi1 panel, the images in Fig. 4G, i and l, corresponding to “smad1 MO” and “smad5 MO + samd1/9 mRNA” samples, respectively, were inadvertently reused during figure preparation. This error has now been corrected using images pertaining to each treatment and sample. This correction does not affect the results or conclusions of the work.jbc;295/52/18650/F4F1F4Figure 4G.