The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.

• MeSH
• Enzyme Activation MeSH
• Genes, cdc MeSH
• Rad52 DNA Repair and Recombination Protein genetics   metabolism   MeSH
• Hydroxyurea metabolism   MeSH
• Nucleic Acid Synthesis Inhibitors metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• DNA Repair * MeSH
• DNA Damage MeSH
• Protein Kinases genetics   metabolism   MeSH
• Rad51 Recombinase metabolism   MeSH
• DNA Replication * MeSH
• Replication Protein A genetics   metabolism   MeSH
• Signal Transduction * physiology   MeSH
• Check Tag
• Humans MeSH

Východiska: Léčba lokálně pokročilého karcinomu rekta standardně zahrnuje neoadjuvantní chemoradioterapii, po které nastupuje operace s případnou adjuvantní chemoterapií. Kromě neoadjuvantní chemoradioterapie se stále více uplatňuje tzv. totální neoadjuvantní terapie (TNT) spočívající v aplikaci radioterapie a následné kombinované chemoterapie, a to v různých sekvencích a kombinacích. Jejím hlavním přínosem je významné snížení rizika rozvoje vzdálených metastáz a současně zlepšení lokální kontroly, které může v indikovaných případech umožnit non-operativní management. Pacienti s nádory s mikrosatelitní instabilitou a deficientním systémem mismatch repair (MSI/dMMR) se začínají při zvažování TNT vyčleňovat jako samostatná skupina profitující z jiného léčebného přístupu. Případ: Náš případ popisuje diagnostiku a léčbu pacientky s lokálně pokročilým karcinomem rekta, která byla pro klinické charakteristiky onemocnění indikovaná k prediktivnímu molekulárně genetickému vyšetření, jež potvrdilo mikrosatelitní instabilitu nádoru. Na základě tohoto nálezu byla u pacientky po iniciální krátké radioterapii místo chemoterapie indikována neoadjuvatní imunoterapie. Následná operace potvrdila kompletní patologickou odpověď. Pacientka absolvovala tuto léčbu s dobrou tolerancí, je nyní v trvající kompletní remisi a dále pečlivě dispenzarizována dle doporučených postupů. Závěr: Molekulárně genetické testování má u karcinomu rekta zásadní význam a mělo by být vstupně provedeno u všech pokročilejších nádorů, u kterých se kromě operace zvažuje jakákoli jiná možná onkologická léčba. Nález MSI/dMMR vyžaduje specifický léčebný přístup, který přináší pacientům s těmito nádory maximální benefit.

Background: Typically, the management of locally advanced rectal cancer consists of neoadjuvant chemoradiotherapy, followed by surgery and adjuvant chemotherapy. In addition to neoadjuvant chemoradiotherapy, total neoadjuvant therapy (TNT) involving radiotherapy and combined chemotherapy has been increasingly used and shown to reduce the risk of distant metastasis and improve local control. Patients with microsatellite instability and deficient mismatch repair (MSI/dMMR) tumors represent a specific group that benefits from different approaches if TNT is considered. Case: Our case report describes the diagnosis and treatment of a patient with locally advanced rectal cancer indicated for clinical characteristics to predictive molecular testing. Microsatellite instability was confirmed. Based on this finding, after short-course radiotherapy, she was offered neoadjuvant immunotherapy with checkpoint inhibitors. She subsequently underwent surgery with a confirmed pathologic complete response. The treatment was well-tolerated and she stays in complete remission, with a follow-up according to the standard recommendations. Conclusion: This case highlights the importance of molecular testing in rectal cancer, which should be performed in all advanced cases requiring more intensive oncologic therapy than surgery alone. MSI/dMMR status indicates the need for a specific approach that may significantly improve the outcomes of these patients.

• MeSH
• Adult MeSH
• Immune Checkpoint Inhibitors * pharmacology   therapeutic use   MeSH
• Humans MeSH
• Microsatellite Instability MeSH
• Rectal Neoplasms * drug therapy   genetics   pathology   MeSH
• Neoadjuvant Therapy methods   MeSH
• DNA Mismatch Repair genetics   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. We also show that tumour-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and that cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumour suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.

• MeSH
• Alleles MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Cell Line MeSH
• Cell Cycle * genetics   radiation effects   MeSH
• Checkpoint Kinase 2 MeSH
• DNA-Binding Proteins MeSH
• cdc25 Phosphatases * physiology   radiation effects   MeSH
• Phosphorylation MeSH
• Radiation, Ionizing MeSH
• Humans MeSH
• Mice MeSH
• Tumor Suppressor Proteins MeSH
• Protein Serine-Threonine Kinases * physiology   MeSH
• Protein Kinases physiology   genetics   MeSH
• Cell Cycle Proteins MeSH
• DNA Replication * radiation effects   MeSH
• S Phase radiation effects   MeSH
• Serine metabolism   MeSH
• Signal Transduction MeSH
• Radiation Tolerance MeSH
• Transfection MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.

• MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Checkpoint Kinase 2 MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• DNA-Binding Proteins metabolism   MeSH
• Phosphorylation MeSH
• Histones metabolism   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• DNA, Single-Stranded metabolism   MeSH
• Caffeine pharmacology   MeSH
• Humans MeSH
• RNA, Small Interfering pharmacology   genetics   MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• DNA Damage * physiology   MeSH
• Protein Serine-Threonine Kinases physiology   drug effects   MeSH
• Protein Kinases pharmacology   physiology   genetics   MeSH
• Cell Cycle Proteins physiology   genetics   metabolism   MeSH
• Purines pharmacology   MeSH
• DNA Replication physiology   drug effects   MeSH
• Replication Protein A MeSH
• Staurosporine * analogs & derivatives   pharmacology   MeSH
• Check Tag
• Humans MeSH

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

• MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Phosphorylation MeSH
• HCT116 Cells MeSH
• G1 Phase Cell Cycle Checkpoints genetics   physiology   MeSH
• Humans MeSH
• DNA Repair genetics   physiology   MeSH
• DNA Replication genetics   physiology   MeSH
• RNA Polymerase II genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion-deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10-100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in 'immunologically cold' tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy.

• MeSH
• Survival Analysis MeSH
• B7-H1 Antigen antagonists & inhibitors   MeSH
• Child MeSH
• Adult MeSH
• Immune Checkpoint Inhibitors pharmacology   therapeutic use   MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Biomarkers, Tumor MeSH
• Tumor Microenvironment MeSH
• Neoplasms drug therapy   MeSH
• DNA Repair genetics   MeSH
• Prospective Studies MeSH
• DNA Replication genetics   MeSH
• Retrospective Studies MeSH
• Germ-Line Mutation * MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Observational Study MeSH
• Research Support, Non-U.S. Gov't MeSH

DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.

• MeSH
• Chromatin MeSH
• DNA genetics   MeSH
• Emetine * pharmacology   MeSH
• DNA, Single-Stranded * MeSH
• DNA Replication MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

To preserve genetic integrity, mammalian cells exposed to ionizing radiation activate the ATM kinase, which initiates a complex response-including the S-phase checkpoint pathways-to delay DNA replication. Defects in ATM or its substrates Nbs1 or Chk2 (ref. 3), the Nbs1-interacting Mre11 protein, or the Chk2-regulated Cdc25A-Cdk2 cascade all cause radio-resistant DNA synthesis (RDS). It is unknown, however, whether these proteins operate in a common signaling cascade. Here we show that experimental blockade of either the Nbs1-Mre11 function or the Chk2-triggered events leads to a partial RDS phenotype in human cells. In contrast, concomitant interference with Nbs1-Mre11 and the Chk2-Cdc25A-Cdk2 pathways entirely abolishes inhibition of DNA synthesis induced by ionizing radiation, resulting in complete RDS analogous to that caused by defective ATM. In addition, Cdk2-dependent loading of Cdc45 onto replication origins, a prerequisite for recruitment of DNA polymerase, was prevented upon irradiation of normal or Nbs1/Mre11-defective cells but not cells with defective ATM. We conclude that in response to ionizing radiation, phosphorylations of Nbs1 and Chk2 by ATM trigger two parallel branches of the DNA damage-dependent S-phase checkpoint that cooperate by inhibiting distinct steps of DNA replication.

• MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Cell Line MeSH
• Checkpoint Kinase 2 MeSH
• Cyclin-Dependent Kinase 2 MeSH
• Cyclin-Dependent Kinases metabolism   MeSH
• DNA-Binding Proteins MeSH
• cdc25 Phosphatases metabolism   MeSH
• Radiation, Ionizing MeSH
• Nuclear Proteins metabolism   MeSH
• CDC2-CDC28 Kinases * MeSH
• Humans MeSH
• Tumor Suppressor Proteins MeSH
• DNA Damage * MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Protein Kinases metabolism   MeSH
• Cell Cycle Proteins MeSH
• S Phase * physiology   MeSH
• Check Tag
• Humans MeSH

DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition.

• MeSH
• RecQ Helicases physiology   MeSH
• Humans MeSH
• Neoplasms etiology   MeSH
• Genomic Instability MeSH
• DNA Replication * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.

• MeSH
• Enzyme Activation MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Models, Biological MeSH
• Cell Cycle * physiology   radiation effects   MeSH
• Checkpoint Kinase 2 MeSH
• DNA-Binding Proteins MeSH
• cdc25 Phosphatases * physiology   radiation effects   MeSH
• Phosphorylation MeSH
• HeLa Cells MeSH
• Radiation, Ionizing MeSH
• Kinetics MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Proteins MeSH
• Protein Serine-Threonine Kinases physiology   MeSH
• Protein Kinases * metabolism   MeSH
• Cell Cycle Proteins MeSH
• DNA Replication radiation effects   MeSH
• S Phase radiation effects   MeSH
• Serine metabolism   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH