The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage. Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA. Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer. Crowther Audrey audrey.crowther@huskers.unl.edu University of Nebraska-Lincoln 2016-08-28T19:07:05Z 2017-02-10T13:29:15Z

Ataxia Telangiectasia Mutated

UniProt ID: Q13315

Gene Name: ATM

Gene ID: 472

 2016-12-07T12:24:10Z

Checkpoint Recovery Process

 2016-11-09T09:54:23Z

Cellular tumor antigen p53

Gene Name: TP53

UniProt ID: P04637

NCBI Gene ID: 7157

 2016-12-19T10:17:29Z

Gene Name: ATR

Ataxia Telangiectasia and Rad3-Related checkpoint protein

UniProt ID: Q13535

Gene ID: 545

 2016-08-28T22:44:31Z

Homologous Recombination repair

This node represents the alternative Homologous Recombination Repair Pathway

 2017-02-10T13:17:43Z

FA core complex

 2016-12-07T12:24:10Z

This node represents the proteins that act in the Homologous Recombination Repair pathway, including FANCD1N, RAD51, HRR, ssDNARPA, FANCJMLH1, MRN, and BRCA1.

 2016-12-19T09:02:15Z

DNA adducts

 2016-08-28T22:54:12Z

Double-strand break

 2017-02-10T13:17:43Z

Proliferation cell nuclear antigen (PCNA), translesion synthesis (TLS)

Abbreviated from PCNATLS to TLS

UniProt AccessionID: P12004

 2017-02-10T13:17:43Z

Nuclease mediated ICL incision

 2016-12-19T10:17:29Z

Interstrand cross links

 2017-02-10T13:02:30Z

Nuclease mediated ICL incision

 2017-02-10T13:17:43Z

Fanconi anemia group I protein/ Fanconi anemia group D2 protein

Gene Name: FANCI/FANCD2

UniProt ID: Q9NVI1/Q9BXW9

Gene ID: 55215/2175

 2016-12-19T09:02:15Z

DNA End Joining Repair protein

For more info: http://www.ncbi.nlm.nih.gov/mesh/?term=nhej

 2017-02-10T13:02:30Z

ATR activates ATM.

DSB activates ATM.

CHKREC inhibits ATM.

FAcore inhibits ATM.

CHKREC activation leads to ATM dephosphorylation.

ATR phosphorylates ATM.

ATM recognizes the nucleosome disruption caused by DSBs.

The FA pathway is activated after ATM inhibition by KU-55933.

 S_4 1 S_2 1 S_6 1 S_9 1 S_2 1 S_6 1

TLS, NHEJ, HRR2, or FAHRR activate sCHKREC.

CHKREC inactivates itself.

DSB inhbits CHKREC.

ADD inhibits CHKREC.

ICL inhibits CHKREC.

The HRR pathway repairs DNA damage, which activates CHKREC.

The NHEJ pathway repairs DNA damage, which activates CHKREC.

CHKREC inactivates itself (Inferred interaction).

The absence of DSB allows for CHKREC activation.

The HRR pathway repairs DNA damage, which activates CHKREC.

The absence of ICL activates CHKREC (Inferred interaction).

TLS allows for DNA replication to continue past the point of damage, which activates CHKREC.

The absence of ADD activates CHKREC (Inferred interaction).

 S_10 1 S_9 1 S_7 1 S_9 1 S_15 1 S_9 1 S_5 1 S_9 1 S_5 1 S_15 1 S_2 1 S_9 1 S_7 1 S_12 1 S_10 1 S_8 1

NHEJ, ATM, or ATR activates p53.

CHKREC inhibits p53.

CHKREC leads to the dephosphorylation of p53.

NHEJ positively regulates p53 through phosphorylation.

ATM positively regulates p53 through phosphorylation.

ATR positively regulates p53 through phosphorylation.

 S_1 1 S_2 1 S_4 1 S_2 1 S_15 1 S_2 1

CHKREC inhibits ATR.

ATM activates ATR.

ICL activates ATR.

CHKREC inactivates ATR (Inferred interaction).

ATM indirectly activates ATR.

Detection of ICL leads to ATR recuirment to the site of damage via ATRIP.

 S_12 1 S_2 1 S_1 1 S_2 1

NHEJ in conjunction with ICL, DSB, and NUC2 activates HRR2.

FAHRR inhibits HRR2.

NHEJ inhibits HRR2.

TLS in conjunction with DSB and NUC2 activates HRR2.

CHKREC inhibits HRR2.

CHKREC indirectly inhibits HRR2.

NHEJ is a pathway used to repair double-stranded breaks. The activation of NHEJ eliminates the need for the HRR2 pathway.

NHEJ can create DSB, activating HRR2.

NUC2 generates DSBs.

Detection of DSB activates HRR2.

The alternative HRR pathway does not occur if FAHRR is functioning.

Detection of ICL activates HRR2

TLS indirectly activates HRR2.

 S_10 1 S_13 1 S_9 1 S_7 1 S_2 1 S_15 1 S_13 1 S_15 1 S_9 1 S_12 1 S_7 1 S_2 1

ICL in conjunction with ATM or ATR activates FAcore.

CHKREC negatively regulates FAcore.

In mitosis, the FAcore is hyperphosphorylated, which induces its release from FANCM.

ATM recruits proteins involved in the FAcore complex.

ATR recruits proteins involved in the FAcore complex.

Detection of ICL leads to recruitment of the FAcore.

 S_12 1 S_1 1 S_4 1 S_2 1

FANCD2I in conjunction with DSB activates FAHRR.

NHEJ in conjunction with CHKREC inhibits FAHRR.

NHEJ indirectly inhibits FAHRR.

CHKREC indirectly inhibits FAHRR.

Detection of DSB leads to monoubiquitination of FANCD2I, which causes its localized into chromatin-associated foci containing DNA replication and repair factors.

Detection of DSB leads to monoubiquitination of FANCD2I, which causes its localized into chromatin-associated foci containing DNA replication and repair factors.

 S_14 1 S_9 1 S_15 1 S_2 1

NUC1 and NUC2 activate ADD.

TLS inhibits ADD.

NUC1 and NUC2 cleave DNA, creating ADDs.

NUC1 and NUC2 cleave DNA, creating ADDs.

TLS aids in repairing ADDs.

 S_11 1 S_10 1 S_11 1 S_13 1 S_10 1 S_13 1 S_10 1

NUC2 activates DSB.

HRR2 inhibits DSB.

NHEJ inhibits DSB.

FAHRR inhibits DSB.

NUC1 activates DSB.

HRR2 repairs DSBs.

NHEJ repairs DSBs.

NUC1 cleaves DNA, creating DSBs.

FAHRR repairs DSBs.

NUC2 cleaves DNA, creating DSBs.

 S_13 1 S_5 1 S_15 1 S_7 1 S_11 1 S_5 1 S_15 1 S_7 1

ADD activates TLS.

CHKREC inhibits TLS.

FAcore in conjunction with ADD activates TLS.

In the absence of damage, PCNA is SUMOylated to inhibit TLS.

Detection of ADD causes the activation of TLS.

The FAcore channels DNA lesions into damage bypass pathways.

 S_8 1 S_2 1 S_6 1 S_8 1 S_2 1

FANCD21 in conjunction with ICL activates NUC1.

FANCD21 recruits NUC1.

FANCD21 in conjunction with ICL activates NUC1 through unhooking.

 S_14 1 S_12 1

ICL activates itself.

DSB inhibits ICL.

DSB phophorylates ICL, which inhibits ICL.

ICL positively regulates itself (Inferred interaction).

 S_12 1 S_9 1

NUC1 in conjunction with p53 and ICL activates NUC2.

ICL in conjunction with ATM or ATR activates NUC2.

FAcore and FANCD21 activates NUC2.

p53 is predicted to promote nuclease activity.

FANCD21 negatively regulates NUC2 through unhooking.

Nuclease activity has a role in unhooking ICL lesions.

ATM phosphorylates downstream proteins to activate nuclease mediated ICL incision.

ATR phosphorylates downstream proteins to activate nuclease mediated ICL incision.

Nuclease activity has a role in unhooking ICL lesions.

FAcore indirectly negatively regulates NUC2.

 S_11 1 S_3 1 S_12 1 S_6 1 S_14 1 S_12 1 S_1 1 S_4 1 S_6 1 S_14 1

FAcore in conjunction with ATM or ATR, or DSB with ATR or ATM activate FANCD21.

CHKREC inhibits FANCD21.

Upon repair of DNA damage, FANCD2 and FANCI dissociate.

ATM positively regulates FANCD2I through phosphorylation.

ATR positively regulates FANCD2I through phosphorylation.

The presence of DSB promotes FANCD2l to load onto chromatin.

FAcore ubiquitinates FANCD2I.

 S_6 1 S_1 1 S_4 1 S_9 1 S_1 1 S_4 1 S_2 1

DSB in conjuction with NUC2 activates NHEJ.

CHKREC, HRR2, or FAHRR inhibit NHEJ.

When HRR2 is active, NHEJ is not (two different repair mechanisms).

CHKREC inhibits NHEJ through monoubiquiylation.

FAHRR inhibits NHEJ through an interaction with Ku proteins.

NUC2 produces a substrate for NHEJ.

Cleavage of DNA by NUC2 as a result of DSB activates NHEJ.

 S_13 1 S_9 1 S_7 1 S_5 1 S_2 1