The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.
Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.
Pubmed ID: 38554706 RIS Download
Research resources used in this publication
- pBabe TIR1-9myc (RRID:Addgene_47328) (plasmid)
- Lenti‐Cas9‐2A‐Blast (RRID:Addgene_73310) (plasmid)
- MCF-10A (RRID:CVCL_0598) (cell line)
- HCT 116 (RRID:CVCL_0291) (cell line)
- lentiCRISPRv2 hygro (RRID:Addgene_98291) (plasmid)
- lentiCRISPRv2 hygro (RRID:Addgene_98291) (plasmid)
- lentiGuide-Puro (RRID:Addgene_52963) (plasmid)
- lentiGuide-Puro (RRID:Addgene_52963) (plasmid)
- pET15b His-p97 (RRID:Addgene_169020) (plasmid)
- pET15b His-p97 (RRID:Addgene_169020) (plasmid)
- Timeless antibody [EPR5275] (RRID:AB_10863023) (antibody)
- Rabbit anti-SMC3 Antibody, Affinity Purified (RRID:AB_67579) (antibody)
- Rabbit Anti-RPA32 / RPA2 Monoclonal Antibody, Unconjugated, Clone EPR2877Y (RRID:AB_1524336) (antibody)
- Rabbit anti-PSMC2 Antibody, Affinity Purified (RRID:AB_2621268) (antibody)
- PolD2 Antibody (RRID:AB_2620518) (antibody)
- Rabbit anti-PolA1 Antibody, Affinity Purified (RRID:AB_10631612) (antibody)
- Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (RRID:AB_331212) (antibody)
- Rabbit anti-MCM7 Antibody, Affinity Purified (RRID:AB_2034845) (antibody)
- Anti-IL-2 Ralpha, clone 7G7/B6 (RRID:AB_309642) (antibody)
- Anti-Histone H3 Antibody, Unconjugated (RRID:AB_331563) (antibody)
- WAPL (A-7) (RRID:AB_10708870) (antibody)
- SPATA5L1 Antibody (RRID:AB_11029566) (antibody)
- Anti-Ubiquitin Antibody (P4D1) (RRID:AB_628423) (antibody)
- RSL24D1 antibody (RRID:AB_2879950) (antibody)
- SMC1 Antibody (RRID:AB_2192467) (antibody)
- alpha Tubulin (TU-02) (RRID:AB_628408) (antibody)
- Rabbit anti-RFC1 Antibody, Affinity Purified (RRID:AB_2178327) (antibody)
- Rad21 Antibody (RRID:AB_2176615) (antibody)
- Poly/Mono-ADP Ribose (E6F6A) (RRID:AB_2749858) (antibody)
- Mouse Anti-Human POLD3 Monoclonal Antibody, Unconjugated, Clone 3E2 (RRID:AB_606803) (antibody)
- Rabbit anti-PolD1 Antibody, Affinity Purified (RRID:AB_2620353) (antibody)
- PCNA (PC10) Mouse mAb (RRID:AB_2160343) (antibody)
- Anti-phospho-Histone H3 (Ser10) Antibody (RRID:AB_310177) (antibody)
- VCP Antibody (RRID:AB_495511) (antibody)
- RPA2 Antibody (RRID:AB_10002227) (antibody)
- Lamin A antibody (RRID:AB_775965) (antibody)
- BrdU (RRID:AB_10015219) (antibody)
- Anti-HA.11 Epitope Tag (RRID:AB_2820200) (antibody)
- Rabbit Anti-GAPDH Monoclonal Antibody, Unconjugated, Clone 14C10 (RRID:AB_561053) (antibody)
- Chk1 antibody (RRID:AB_2880283) (antibody)
- Calpain 1 Large Subunit (Mu-type) Antibody (RRID:AB_2290836) (antibody)
- Mouse Anti-Human CAPNS1 Monoclonal, Unconjugated, Clone 3c4 (RRID:AB_537367) (antibody)
- Rat Anti-BrdU Monoclonal Antibody, Unconjugated, Clone BU1 / 75 (ICR1) (RRID:AB_305426) (antibody)
- Anti-ATR Antibody (C-1) (RRID:AB_2893291) (antibody)
- Anti-acetyl SMC3 Antibody (Lys105/106), clone 21A7 (RRID:AB_2877126) (antibody)
- Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 (RRID:AB_309864) (antibody)
Additional research tools detected in this publication
None foundAntibodies used in this publication
- HA-Tag (C29F4) Rabbit mAb (RRID:AB_1549585)
- PARP (46D11) Rabbit mAb (RRID:AB_659884)
- Timeless antibody [EPR5275] (RRID:AB_10863023)
- Rabbit anti-SMC3 Antibody, Affinity Purified (RRID:AB_67579)
- Rabbit Anti-RPA32 / RPA2 Monoclonal Antibody, Unconjugated, Clone EPR2877Y (RRID:AB_1524336)
- PolD2 Antibody (RRID:AB_2620518)
- Rabbit anti-PSMC2 Antibody, Affinity Purified (RRID:AB_2621268)
- Rabbit anti-PolA1 Antibody, Affinity Purified (RRID:AB_10631612)
- Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (RRID:AB_331212)
- Rabbit anti-MCM7 Antibody, Affinity Purified (RRID:AB_2034845)
- Anti-IL-2 Ralpha, clone 7G7/B6 (RRID:AB_309642)
- WAPL (A-7) (RRID:AB_10708870)
- Anti-Histone H3 Antibody, Unconjugated (RRID:AB_331563)
- alpha Tubulin (TU-02) (RRID:AB_628408)
- SPATA5L1 Antibody (RRID:AB_11029566)
- RSL24D1 antibody (RRID:AB_2879950)
- Anti-Ubiquitin Antibody (P4D1) (RRID:AB_628423)
- Rabbit anti-RFC1 Antibody, Affinity Purified (RRID:AB_2178327)
- SMC1 Antibody (RRID:AB_2192467)
- Rad21 Antibody (RRID:AB_2176615)
- Poly/Mono-ADP Ribose (E6F6A) (RRID:AB_2749858)
- Rabbit anti-PolD1 Antibody, Affinity Purified (RRID:AB_2620353)
- Mouse Anti-Human POLD3 Monoclonal Antibody, Unconjugated, Clone 3E2 (RRID:AB_606803)
- PCNA (PC10) Mouse mAb (RRID:AB_2160343)
- RPA2 Antibody (RRID:AB_10002227)
- Anti-phospho-Histone H3 (Ser10) Antibody (RRID:AB_310177)
- VCP Antibody (RRID:AB_495511)
- Lamin A antibody (RRID:AB_775965)
- BrdU (RRID:AB_10015219)
- Rabbit Anti-GAPDH Monoclonal Antibody, Unconjugated, Clone 14C10 (RRID:AB_561053)
- Anti-HA.11 Epitope Tag (RRID:AB_2820200)
- Mouse Anti-Human CAPNS1 Monoclonal, Unconjugated, Clone 3c4 (RRID:AB_537367)
- Rat Anti-BrdU Monoclonal Antibody, Unconjugated, Clone BU1 / 75 (ICR1) (RRID:AB_305426)
- Chk1 antibody (RRID:AB_2880283)
- Calpain 1 Large Subunit (Mu-type) Antibody (RRID:AB_2290836)
- Anti-ATR Antibody (C-1) (RRID:AB_2893291)
- Anti-acetyl SMC3 Antibody (Lys105/106), clone 21A7 (RRID:AB_2877126)
- Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 (RRID:AB_309864)
Associated grants
- Agency: NCI NIH HHS, United States
Id: R01 CA174904 - Agency: NCI NIH HHS, United States
Id: P01 CA265794 - Agency: NIGMS NIH HHS, United States
Id: R01 GM101149 - Agency: Wellcome Trust, United Kingdom
- Agency: Medical Research Council, United Kingdom
Id: MR/T029471/1
Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.
This is a list of tools and resources that we have found mentioned in this publication.
HA-Tag (C29F4) Rabbit mAb (antibody)
RRID:AB_1549585
This monoclonal targets HA-Tag
View all literature mentionsPARP (46D11) Rabbit mAb (antibody)
RRID:AB_659884
This monoclonal targets PARP
View all literature mentionsTimeless antibody [EPR5275] (antibody)
RRID:AB_10863023
This monoclonal targets Timeless antibody [EPR5275]
View all literature mentionsRabbit anti-SMC3 Antibody, Affinity Purified (antibody)
RRID:AB_67579
This polyclonal targets SMC3
View all literature mentionsRabbit Anti-RPA32 / RPA2 Monoclonal Antibody, Unconjugated, Clone EPR2877Y (antibody)
RRID:AB_1524336
This monoclonal targets RPA32/RPA2
View all literature mentionsRabbit anti-PSMC2 Antibody, Affinity Purified (antibody)
RRID:AB_2621268
This polyclonal targets PSMC2
View all literature mentionsRabbit anti-PolA1 Antibody, Affinity Purified (antibody)
RRID:AB_10631612
This polyclonal targets PolA1
View all literature mentionsPhospho-Chk1 (Ser345) (133D3) Rabbit mAb (antibody)
RRID:AB_331212
This monoclonal targets Chk1, phospho (Ser345)
View all literature mentionsRabbit anti-MCM7 Antibody, Affinity Purified (antibody)
RRID:AB_2034845
This polyclonal targets MCM7
View all literature mentionsAnti-IL-2 Ralpha, clone 7G7/B6 (antibody)
RRID:AB_309642
This monoclonal targets IL-2 Ralpha clone 7G7/B6
View all literature mentionsWAPL (A-7) (antibody)
RRID:AB_10708870
This monoclonal targets WAPL (A-7)
View all literature mentionsAnti-Histone H3 Antibody, Unconjugated (antibody)
RRID:AB_331563
This polyclonal targets Histone H3
View all literature mentionsalpha Tubulin (TU-02) (antibody)
RRID:AB_628408
This monoclonal targets alpha Tubulin (TU-02)
View all literature mentionsSPATA5L1 Antibody (antibody)
RRID:AB_11029566
This polyclonal targets SPATA5L1
View all literature mentionsRSL24D1 antibody (antibody)
RRID:AB_2879950
This polyclonal targets RSL24D1
View all literature mentionsAnti-Ubiquitin Antibody (P4D1) (antibody)
RRID:AB_628423
This monoclonal targets Ubiquitin
View all literature mentionsRabbit anti-RFC1 Antibody, Affinity Purified (antibody)
RRID:AB_2178327
This polyclonal targets RFC1
View all literature mentionsPoly/Mono-ADP Ribose (E6F6A) (antibody)
RRID:AB_2749858
This monoclonal targets poly/mono-ADP ribose
View all literature mentionsRabbit anti-PolD1 Antibody, Affinity Purified (antibody)
RRID:AB_2620353
This polyclonal targets PolD1
View all literature mentionsMouse Anti-Human POLD3 Monoclonal Antibody, Unconjugated, Clone 3E2 (antibody)
RRID:AB_606803
This monoclonal targets Human POLD3
View all literature mentionsPCNA (PC10) Mouse mAb (antibody)
RRID:AB_2160343
This monoclonal targets PCNA
View all literature mentionsAnti-phospho-Histone H3 (Ser10) Antibody (antibody)
RRID:AB_310177
This unknown targets phospho-Histone H3 (Ser10)
View all literature mentionsLamin A antibody (antibody)
RRID:AB_775965
This polyclonal targets Human Lamin A
View all literature mentionsRabbit Anti-GAPDH Monoclonal Antibody, Unconjugated, Clone 14C10 (antibody)
RRID:AB_561053
This recombinant monoclonal targets GAPDH
View all literature mentionsAnti-HA.11 Epitope Tag (antibody)
RRID:AB_2820200
This monoclonal targets HA.11
View all literature mentionsMouse Anti-Human CAPNS1 Monoclonal, Unconjugated, Clone 3c4 (antibody)
RRID:AB_537367
This monoclonal targets Human CAPNS1
View all literature mentionsRat Anti-BrdU Monoclonal Antibody, Unconjugated, Clone BU1 / 75 (ICR1) (antibody)
RRID:AB_305426
This monoclonal targets BrdU
View all literature mentionsCalpain 1 Large Subunit (Mu-type) Antibody (antibody)
RRID:AB_2290836
This polyclonal targets CAPN9
View all literature mentionsAnti-ATR Antibody (C-1) (antibody)
RRID:AB_2893291
This monoclonal targets ATR
View all literature mentionsAnti-acetyl SMC3 Antibody (Lys105/106), clone 21A7 (antibody)
RRID:AB_2877126
This monoclonal targets SMC3
View all literature mentionsAnti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 (antibody)
RRID:AB_309864
This monoclonal targets Histone H2A.X pSer139
View all literature mentionsMCF-10A (cell line)
RRID:CVCL_0598
Cell line MCF-10A is a Spontaneously immortalized cell line with a species of origin Homo sapiens
View all literature mentionsHCT 116 (cell line)
RRID:CVCL_0291
Cell line HCT 116 is a Cancer cell line with a species of origin Homo sapiens (Human)
View all literature mentionsTimeless antibody [EPR5275] (antibody)
RRID:AB_10863023
This monoclonal targets Timeless antibody [EPR5275]
View all literature mentionsRabbit anti-SMC3 Antibody, Affinity Purified (antibody)
RRID:AB_67579
This polyclonal targets SMC3
View all literature mentionsRabbit Anti-RPA32 / RPA2 Monoclonal Antibody, Unconjugated, Clone EPR2877Y (antibody)
RRID:AB_1524336
This monoclonal targets RPA32/RPA2
View all literature mentionsRabbit anti-PSMC2 Antibody, Affinity Purified (antibody)
RRID:AB_2621268
This polyclonal targets PSMC2
View all literature mentionsRabbit anti-PolA1 Antibody, Affinity Purified (antibody)
RRID:AB_10631612
This polyclonal targets PolA1
View all literature mentionsPhospho-Chk1 (Ser345) (133D3) Rabbit mAb (antibody)
RRID:AB_331212
This monoclonal targets Chk1, phospho (Ser345)
View all literature mentionsRabbit anti-MCM7 Antibody, Affinity Purified (antibody)
RRID:AB_2034845
This polyclonal targets MCM7
View all literature mentionsAnti-IL-2 Ralpha, clone 7G7/B6 (antibody)
RRID:AB_309642
This monoclonal targets IL-2 Ralpha clone 7G7/B6
View all literature mentionsAnti-Histone H3 Antibody, Unconjugated (antibody)
RRID:AB_331563
This polyclonal targets Histone H3
View all literature mentionsWAPL (A-7) (antibody)
RRID:AB_10708870
This monoclonal targets WAPL (A-7)
View all literature mentionsSPATA5L1 Antibody (antibody)
RRID:AB_11029566
This polyclonal targets SPATA5L1
View all literature mentionsAnti-Ubiquitin Antibody (P4D1) (antibody)
RRID:AB_628423
This monoclonal targets Ubiquitin
View all literature mentionsRSL24D1 antibody (antibody)
RRID:AB_2879950
This polyclonal targets RSL24D1
View all literature mentionsalpha Tubulin (TU-02) (antibody)
RRID:AB_628408
This monoclonal targets alpha Tubulin (TU-02)
View all literature mentionsRabbit anti-RFC1 Antibody, Affinity Purified (antibody)
RRID:AB_2178327
This polyclonal targets RFC1
View all literature mentionsPoly/Mono-ADP Ribose (E6F6A) (antibody)
RRID:AB_2749858
This monoclonal targets poly/mono-ADP ribose
View all literature mentionsMouse Anti-Human POLD3 Monoclonal Antibody, Unconjugated, Clone 3E2 (antibody)
RRID:AB_606803
This monoclonal targets Human POLD3
View all literature mentionsRabbit anti-PolD1 Antibody, Affinity Purified (antibody)
RRID:AB_2620353
This polyclonal targets PolD1
View all literature mentionsPCNA (PC10) Mouse mAb (antibody)
RRID:AB_2160343
This monoclonal targets PCNA
View all literature mentionsAnti-phospho-Histone H3 (Ser10) Antibody (antibody)
RRID:AB_310177
This unknown targets phospho-Histone H3 (Ser10)
View all literature mentionsLamin A antibody (antibody)
RRID:AB_775965
This polyclonal targets Human Lamin A
View all literature mentionsAnti-HA.11 Epitope Tag (antibody)
RRID:AB_2820200
This monoclonal targets HA.11
View all literature mentionsRabbit Anti-GAPDH Monoclonal Antibody, Unconjugated, Clone 14C10 (antibody)
RRID:AB_561053
This recombinant monoclonal targets GAPDH
View all literature mentionsCalpain 1 Large Subunit (Mu-type) Antibody (antibody)
RRID:AB_2290836
This polyclonal targets CAPN9
View all literature mentionsMouse Anti-Human CAPNS1 Monoclonal, Unconjugated, Clone 3c4 (antibody)
RRID:AB_537367
This monoclonal targets Human CAPNS1
View all literature mentionsRat Anti-BrdU Monoclonal Antibody, Unconjugated, Clone BU1 / 75 (ICR1) (antibody)
RRID:AB_305426
This monoclonal targets BrdU
View all literature mentionsAnti-ATR Antibody (C-1) (antibody)
RRID:AB_2893291
This monoclonal targets ATR
View all literature mentionsAnti-acetyl SMC3 Antibody (Lys105/106), clone 21A7 (antibody)
RRID:AB_2877126
This monoclonal targets SMC3
View all literature mentionsAnti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 (antibody)
RRID:AB_309864
This monoclonal targets Histone H2A.X pSer139
View all literature mentions
