The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

Alver, Robert C.; Zhang, Tianji; Josephrajan, Ajeetha; Fultz, Brandy L.; Hendrix, Chance J.; Das-Bradoo, Sapna; Bielinsky, Anja‐Katrin

doi:10.1093/nar/gku479

Cited by 25 publications

(34 citation statements)

References 51 publications

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“…1B, left panel). When the same bait vector and yeast strain were used in a previous report by others, a similar extra band was detected by the antiMyc mAb (32).…”

Section: Fpn1 Interacts With Pcbp2 But Not With Pcbp1 Pcbp3 or Pcbpsupporting

confidence: 68%

“…Considering this finding, two other sources of anti-HA mAbs (from Wako Chemicals (Osaka Japan) and Covance Laboratories (Madison, WI)) were utilized for detection, and again, a similar pattern of multiple bands was detected (data not shown). Of interest, when the same prey vector and yeast strain were used in previous reports by others, similar multiple bands were detected by the anti-HA mAb (32,33).…”

Section: Fpn1 Interacts With Pcbp2 But Not With Pcbp1 Pcbp3 or Pcbpsupporting

confidence: 67%

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Iron Export through the Transporter Ferroportin 1 Is Modulated by the Iron Chaperone PCBP2

Izumi

Richardson

Imada

et al. 2016

Journal of Biological Chemistry

127

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Ferroportin 1 (FPN1) is an iron export protein found in mammals. FPN1 is important for the export of iron across the basolateral membrane of absorptive enterocytes and across the plasma membrane of macrophages. The expression of FPN1 is regulated by hepcidin, which binds to FPN1 and then induces its degradation. Previously, we demonstrated that divalent metal transporter 1 (DMT1) interacts with the intracellular iron chaperone protein poly(rC)-binding protein 2 (PCBP2). Subsequently, PCBP2 receives iron from DMT1 and then disengages from the transporter. In this study, we investigated the function of PCBP2 in iron export. Mammalian genomes encode four PCBPs (i.e. PCBP1-4). Here, for the first time, we demonstrated using both yeast and mammalian cells that PCBP2, but not PCBP1, PCBP3, or PCBP4, binds with FPN1. Importantly, ironloaded, but not iron-depleted, PCBP2 interacts with FPN1. The PCBP2-binding domain of FPN1 was identified in its C-terminal cytoplasmic region. The silencing of PCBP2 expression suppressed FPN1-dependent iron export from cells. These results suggest that FPN1 exports iron received from the iron chaperone PCBP2. Therefore, it was found that PCBP2 modulates cellular iron export, which is an important physiological process.Iron is an essential but potentially hazardous bio-metal (1). Because of its ability to readily accept or donate electrons, iron is a valuable cofactor that is used in oxygen transport, electron transfer, and DNA synthesis (2, 3). However, iron is potentially toxic because it catalyzes the generation of reactive oxygen species. The ensuing oxidative stress is associated with damage to cellular molecules, injury to tissues, and disease via processes including hydroxyl radical formation, glutathione depletion, protein aggregation, lipid peroxidation, and nucleic acid modification (4, 5).Despite its high abundance in nature, ferric iron is poorly bioavailable due to its exceedingly low solubility at physiological pH. Thus, the acquisition and usage of iron presents a considerable challenge to cells and organisms, which have evolved sophisticated mechanisms to satisfy their metabolic needs and concomitantly minimize the risk of toxicity.It has long been known that there is very little, yet potentially toxic, low-molecular-weight iron in rapidly metabolizing cells (6). However, the molecular mechanisms involved in intracellular iron transport have remained elusive (7). Recently, it was revealed that one of the mechanisms of iron transport and metabolism involves intracellular chaperone proteins (8). It was reported that poly(rC)-binding protein 1 (PCBP1), 3 also referred to as ␣-CP1 or hnRNP E1, is a cytosolic iron chaperone protein that delivers iron to ferritin (9). PCBP1 binds to iron with micromolar affinity at a molar ratio of 3:1 Fe:PCBP1. Notably, PCBP1 has been shown previously to function as an RNAand DNA-binding protein and has been identified as a member of a family of four homologous proteins containing three heterogeneous nuclear ribonucleoprotein K-homology (KH) do...

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“…1B, left panel). When the same bait vector and yeast strain were used in a previous report by others, a similar extra band was detected by the antiMyc mAb (32).…”

Section: Fpn1 Interacts With Pcbp2 But Not With Pcbp1 Pcbp3 or Pcbpsupporting

confidence: 68%

Section: Fpn1 Interacts With Pcbp2 But Not With Pcbp1 Pcbp3 or Pcbpsupporting

confidence: 67%

Iron Export through the Transporter Ferroportin 1 Is Modulated by the Iron Chaperone PCBP2

Izumi

Richardson

Imada

et al. 2016

Journal of Biological Chemistry

127

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“…Alver et al 33 . have shown that N-terminus of Mcm10 plays an important role in resisting the DNA damage induced by different genomic insults including treatment with camptothecin in budding yeast.…”

Section: Resultsmentioning

confidence: 99%

Xenopus Mcm10 is a CDK-substrate required for replication fork stability

et al. 2016

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During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in Xenopus egg extracts. We show that Xenopus Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress.

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“…All transgenes were expressed from a plasmid that contained the endogenous MCM10 promoter [17]. Plasmids expressing wild-type or mutant MCM10 were transformed into the mcm10-1 strain and spotted on SC-URA plates in 10-fold dilutions.…”

Section: Resultsmentioning

confidence: 99%

Mapping ubiquitination sites of S. cerevisiae Mcm10

Zhang¹,

Fultz²,

Das-Bradoo³

et al. 2016

Biochemistry and Biophysics Reports

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Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.

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The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

Cited by 25 publications

References 51 publications

Iron Export through the Transporter Ferroportin 1 Is Modulated by the Iron Chaperone PCBP2

Iron Export through the Transporter Ferroportin 1 Is Modulated by the Iron Chaperone PCBP2

Xenopus Mcm10 is a CDK-substrate required for replication fork stability

Mapping ubiquitination sites of S. cerevisiae Mcm10

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