Note: Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse
Note for: Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse
(doi: 10.1128/MCB.02043-06)
Fbh1 (F-box DNA helicase 1) -- treating with the DNA damaging agents; no prominent phenotype, contradict with yeast -- there might be the compensate pw in DT40.
FBH(-/-) phenotype in DT40;
1.increase sister chromatid exchange
2.formation of radial structures
3.prevent extensive strand exchange during HR
4.work with Bloom helicase - controling recombination-mediated DSB repair at replication block
5.work with Bloom helicase - reduce frequency of crossover
Two major pw to release the replication block;
1. TLS -- using TLS polymerase, synthesis directly across the DNA damage
2. HR -- transient template switch from the damage to newly synthesized daughter strands
There has been proposed that db holliday junctions are dissolved by concerted action of RecQ helicase, Blm and Topo-IIIalpha
RecQ family helicases have been implicated in the initiation of replication restart without crossover and resolution of recombination intermediates, their function remains poorly understood even in yeast due to the complexity of repair reactions following replication block and the lack of an appropriate phenotypic assay to monitor the late steps of HR in vivo.
Substrate for Fbh1 cannot yet been found.
In conclusion - FBH1 acts in parallel with Blm and prevent the strand exchange during the recombination initiated by stalled replication forks. Reducing frequency of crossover.
chicken FBH1 - 1012 aa;
55% - human
50% - mouse
26% - yeast
in aa identity
FBH1 - shows same growth kinetics and cell cycle distribution as wild-type.
Evaluation of Fbh1 on HR phenotype;
1. DNA damaging agent
2. gene-targeting frequency
3. HR-mediated repair by I-SceI restriction enzyme
4. HR-dependent diversification of Ig gene (Ig gene conversion)
5. Foci formation
Result -- all HR phenotypes showed the same as WT.
but Fbh1 KO showed increase of spontaneous SCE; there are two possibilities have been rasied by this acttivity -
1.increase in endogenous DNA damage which at the end stimulate the SCE
2.elevated ratio of crossover-type HR relative to non-crossover-type HR
The team tested further with CPT (DBS-when replicate) and UV (SS gaps-during replication) (which does not show the sensitivity before).
Higher elevation of SCE - explore more on functional interactions between Fbh1 + other genes; FancC/Rad18.
Given that the SCE phenotype of FBH-KO cells suggested that recombination events more frequently resulted in crossover, we sought to test the relationship between Fbh1 and the core recombination factor Rad54.
Because of there is much higher of DSB in both sister chromatids with the same location in DB-KO (Fbh1+Rad54) - team raised the possibility to explain this phenomenon; First, both Fbh1 and Rad54 may be involved in the same stage of HR, with Fbh1 acting as a backup for Rad54-dependent HR. Alternatively, Fbh1 may prevent the formation of abnormal recombination intermediates, which lead to chromosome-type breaks in the absence of Rad54.
These results suggest a possible involvement of the Fbh1 helicase, in parallel with the Blm helicase, in resolving otherwise toxic recombination intermediates produced at the sites of stalled replication forks.
there are some processes to restrict the extent of strand exchange during recombination produced at the sites of stalled replication forks, resulting in a reduced frequency of crossover formation.
Precise process of crossover remains to be determined.
Fbh1 being involved in preventing the formation of recombination intermediates following replication blockage.
Somatic cell division in vertebrates;
- non-crossover-type HR
Fbh1 is likely to prevent extensive exchange between two homologous sequences, thereby facilitating HR dependent repair of single-ended breaks formed during replication.
Thus, the absence of Fbh1 or Blm may cause an accumulation of recombination intermediates that normally require these DNA helicases for their resolution.
we conclude that the Fbh1-dependent pathway is likely to act in parallel to Blm in cellular tolerance of single-ended breaks resulting from replication blocks.
No human FBH1 mutants have been reported, possibly because FBH1 disruption alone does not result in a clinically detectable syndrome or, although less likely given the lack of a phenotype in DT40 cells, is lethal. However, it will be interesting to determine whether a defect in this helicase function is associated with cancer predisposition or even a genetic disease akin to Bloom’s and Werner’s syndromes.
(doi: 10.1128/MCB.02043-06)
Fbh1 (F-box DNA helicase 1) -- treating with the DNA damaging agents; no prominent phenotype, contradict with yeast -- there might be the compensate pw in DT40.
FBH(-/-) phenotype in DT40;
1.increase sister chromatid exchange
2.formation of radial structures
3.prevent extensive strand exchange during HR
4.work with Bloom helicase - controling recombination-mediated DSB repair at replication block
5.work with Bloom helicase - reduce frequency of crossover
Two major pw to release the replication block;
1. TLS -- using TLS polymerase, synthesis directly across the DNA damage
2. HR -- transient template switch from the damage to newly synthesized daughter strands
There has been proposed that db holliday junctions are dissolved by concerted action of RecQ helicase, Blm and Topo-IIIalpha
RecQ family helicases have been implicated in the initiation of replication restart without crossover and resolution of recombination intermediates, their function remains poorly understood even in yeast due to the complexity of repair reactions following replication block and the lack of an appropriate phenotypic assay to monitor the late steps of HR in vivo.
Substrate for Fbh1 cannot yet been found.
In conclusion - FBH1 acts in parallel with Blm and prevent the strand exchange during the recombination initiated by stalled replication forks. Reducing frequency of crossover.
chicken FBH1 - 1012 aa;
55% - human
50% - mouse
26% - yeast
in aa identity
FBH1 - shows same growth kinetics and cell cycle distribution as wild-type.
Evaluation of Fbh1 on HR phenotype;
1. DNA damaging agent
2. gene-targeting frequency
3. HR-mediated repair by I-SceI restriction enzyme
4. HR-dependent diversification of Ig gene (Ig gene conversion)
5. Foci formation
Result -- all HR phenotypes showed the same as WT.
but Fbh1 KO showed increase of spontaneous SCE; there are two possibilities have been rasied by this acttivity -
1.increase in endogenous DNA damage which at the end stimulate the SCE
2.elevated ratio of crossover-type HR relative to non-crossover-type HR
The team tested further with CPT (DBS-when replicate) and UV (SS gaps-during replication) (which does not show the sensitivity before).
Higher elevation of SCE - explore more on functional interactions between Fbh1 + other genes; FancC/Rad18.
Given that the SCE phenotype of FBH-KO cells suggested that recombination events more frequently resulted in crossover, we sought to test the relationship between Fbh1 and the core recombination factor Rad54.
Because of there is much higher of DSB in both sister chromatids with the same location in DB-KO (Fbh1+Rad54) - team raised the possibility to explain this phenomenon; First, both Fbh1 and Rad54 may be involved in the same stage of HR, with Fbh1 acting as a backup for Rad54-dependent HR. Alternatively, Fbh1 may prevent the formation of abnormal recombination intermediates, which lead to chromosome-type breaks in the absence of Rad54.
These results suggest a possible involvement of the Fbh1 helicase, in parallel with the Blm helicase, in resolving otherwise toxic recombination intermediates produced at the sites of stalled replication forks.
there are some processes to restrict the extent of strand exchange during recombination produced at the sites of stalled replication forks, resulting in a reduced frequency of crossover formation.
Precise process of crossover remains to be determined.
Fbh1 being involved in preventing the formation of recombination intermediates following replication blockage.
Somatic cell division in vertebrates;
- non-crossover-type HR
Fbh1 is likely to prevent extensive exchange between two homologous sequences, thereby facilitating HR dependent repair of single-ended breaks formed during replication.
Thus, the absence of Fbh1 or Blm may cause an accumulation of recombination intermediates that normally require these DNA helicases for their resolution.
we conclude that the Fbh1-dependent pathway is likely to act in parallel to Blm in cellular tolerance of single-ended breaks resulting from replication blocks.
No human FBH1 mutants have been reported, possibly because FBH1 disruption alone does not result in a clinically detectable syndrome or, although less likely given the lack of a phenotype in DT40 cells, is lethal. However, it will be interesting to determine whether a defect in this helicase function is associated with cancer predisposition or even a genetic disease akin to Bloom’s and Werner’s syndromes.
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