Crystal Structure of the Human Primase

Baranovskiy, Andrey G.; Zhang, Yinbo; Suwa, Yuichi; Babayeva, Nigar D.; Gu, Jianyou; Pavlov, Youri I.; Tahirov, T.H.

doi:10.1074/jbc.m114.624742

Cited by 73 publications

(118 citation statements)

References 51 publications

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“…1 and 2). These findings help to explain why in previous primer extension studies, where the substrate was missing TriP and sometimes also the 3Ј-overhang, the effect of p58 C deletion was not so prominent, and its significance for primer elongation was underestimated (13,18,33). Earlier, Podust et al (34) compared the activity of Prim-Pol␣ purified from the human placenta on ribooligoadenylates of varying length (from 2 to 10 nucleotides) and found that deletion of the ␤-and ␥-phosphates from the 5Ј-terminus of RNA primers significantly diminishes their extension.…”

Section: P58 C Plays An Important Role In Primer Extension and Lengthmentioning

confidence: 67%

“…Cloning, Expression, and Purification-Cloning, expression, and purification to homogeneity of the human primase het-erodimer (p49-p58; p49 contains 420 amino acids, p58 contains 509), the C-terminal domain of p58 (p58 C ; amino acids 266 -456), primase without p58 C (p49-p58 N ; p58 N contains amino acids , and the human Pol␣ without the catalytic core (p70-p180 C ; p180 C contains amino acids 1265-1462) have been described elsewhere (13,20,21). The complexes p49-p58-p70-p180 C (Prim-Pol␣⌬cat) and p49-p58 N -p70-p180 C (Prim⌬FeS-Pol␣⌬cat) have been obtained in the same way as Prim-Pol␣ (6); the pure primase and Pol␣ samples were mixed at an equimolar ratio and incubated on ice for 30 min, and the reconstituted complexes were purified by gel-filtration on a Superose 12 column (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…The large subunit of human primase is composed of two separate domains connected with a long linker, which indicates a significant conformational flexibility of this subunit (13). The N-terminal domain (p58 N ) provides a platform for interactions with p49 and Pol␣ (6,12) and also plays some role in the orientation of p58 C relative to p49.…”

mentioning

confidence: 99%

“…Biochemical data indicate that p58 C contributes to primase activity and participates in substrate binding by an unknown mechanism (16 -19). Our recent report provided several pieces of evidence that p58 C and p49 cooperate in a cis orientation during primer synthesis, meaning that the template-primer is handled by two functional domains located on the same twosubunit molecule (13).…”

mentioning

confidence: 99%

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Insight into the Human DNA Primase Interaction with Template-Primer

Baranovskiy¹,

Zhang

Suwa³

et al. 2016

Journal of Biological Chemistry

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DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58 C ) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58 C . The specific mode of interaction with a template-primer involving the terminal 5-triphosphate of RNA and the 3-overhang of DNA results in a stable complex between p58 C and the DNA/RNA duplex. Our results explain how p58 C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58 C . These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.The four-subunit primase-polymerase ␣ (Prim-Pol␣) 3 complex possessing DNA primase and DNA polymerase active sites is important for genome replication in eukaryotes (1, 2). PrimPol␣ synthesizes the chimeric RNA-DNA primers for replicative DNA polymerases ⑀ and ␦ (3, 4). In humans, the primase heterodimer contains a small catalytic subunit (p49; also known as p48, PRIM1, Pri1, and PriS) and a large regulatory subunit (p58; also known as PRIM2, Pri2, and PriL). Pol␣ is composed of a large catalytic subunit (p180) and a small accessory subunit (p70). p58 and p70 are connected with p180 through the interaction with a small C-terminal domain (p180 C ) that defines the tight coordination of the RNA-and DNA-polymerizing activities (5-7).Eukaryotic primases have a minimal specific recognition site on DNA and only require a pyrimidine to template the 5Ј-terminal nucleotide of the primer (8, 9). RNA primer synthesis begins with a rate-limiting initiation step that includes binding of the DNA template and two NTPs followed by dinucleotide synthesis (10). Subsequently, primase elongates the generated dinucleotide to the unit-length primer (8 -10-mer) and terminates synthesis. This unique counting ability of DNA primases, which results in RNA primers that are optimal for extension by Pol␣, has a complex mechanism that is currently unclear. Recent structural data revealed that the primase active site located on p49 uses the common mechanism of nucleic acids synthesis, where the catalytic aspartates coordinate two divalent ions and the triphosphate moiety of the incoming NTP (11,12).The large subunit of human primase is composed of two separate domains connected with a long linker, which indicates a significant conformational flexibility of this subunit (13). The N-terminal domain (p58 N ) provides a platform for interactions with p49 and P...

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Section: P58 C Plays An Important Role In Primer Extension and Lengthmentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Insight into the Human DNA Primase Interaction with Template-Primer

Baranovskiy¹,

Zhang

Suwa³

et al. 2016

Journal of Biological Chemistry

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100

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“…We recently solved the crystal structures of human DNA primase and the Pol ␣ catalytic core domain (55,56). Now we have the crystal structures covering all the functional domains of human Prim-Pol ␣ that are necessary for building a model of the entire tetrameric complex.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

Suwa¹,

Gu²,

Baranovskiy³

et al. 2015

Journal of Biological Chemistry

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Background: DNA polymerase ␣ (Pol ␣) initiates DNA synthesis and is indispensable for genome replication.Results: We present a crystal structure of human Pol ␣ minus the catalytic core at 2.5 Å resolution. Conclusion:The mode of interaction between the Pol ␣ subunits is evolutionarily conserved. Significance: The data provide structural insight into the function of the primase-Pol ␣ complex.

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DNA primase polypeptide 1 (PRIM1) involves in estrogen‐induced breast cancer formation through activation of the G2/M cell cycle checkpoint

Lee

Chen

Chia‐Jung

et al. 2018

Intl Journal of Cancer

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The DNA primase polypeptide 1 (PRIM1) is responsible for synthesizing small RNA primers for Okazaki fragments generated during discontinuous DNA replication. PRIM1 mRNA expression levels in breast tumor samples were detected by real-time PCR analysis. Xenografted tumor model was established to study the carcinogenic role of PRIM1 and its potential therapeutic applications. The average PRIM1 mRNA (copy number × 10 /μg) expression was 4.7-fold higher in tumors than in normal tissue (*p = 0.005, n = 254). PRIM1 was detected preferentially at a higher level (>40-fold) in poorly differentiated tumor tissues (n = 46) compared with more highly differentiated tumors tissues (n = 10) (*p = 0.005). Poor overall survival rate was correlated to the estrogen receptor positive (ER+, n = 20) patients with higher PRIM1 expression when compare to the ER- (n = 10) patients (Chi Square test, p = 0.03). Stable expression of PRIM1-siRNA in the ER+ BT-474 cells-xenograft tumors significantly reduced tumor volume in SCID mice (*p = 0.005). The anti-tumoral effects of inotilone isolated from Phellinus linteus was tested and had significant effects on the inhibition of PRIM1 protein expression in ER+ breast cancer cells. In vivo study was performed by administering inotilone (10 mg/kg, twice a week for 6 weeks), which resulted in significantly reduced BT-474-xenografted tumor growth volume compared with control (n =5 per group, *p < 0.05). This study provides evidences for the prognostic effects of PRIM1 with poor overall survival rate in the ER+ patients and will be valuable to test for therapeutic purpose.

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Crystal Structure of the Human Primase

Cited by 73 publications

References 51 publications

Insight into the Human DNA Primase Interaction with Template-Primer

Insight into the Human DNA Primase Interaction with Template-Primer

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

DNA primase polypeptide 1 (PRIM1) involves in estrogen‐induced breast cancer formation through activation of the G2/M cell cycle checkpoint

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