Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site

Juarez-Quintero, Víctor; Peralta-Castro, Antolín; Cardoza, Claudia Guadalupe Benítez; Ellenberger, Tom; Brieba, Luis G.

doi:10.1042/bcj20200922

Cited by 9 publications

(9 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, the exonuclease domain hosts an insertion between helices α4 and α5, which is however also present in T7 DNA polymerase. It is interesting to note that a recent article mentioned this region as probably implicated in shuttling the DNA primer's 3′-end between the pol and exo sites ( 47 ) (see Discussion and PDB 2AJQ).…”

Section: Resultsmentioning

confidence: 99%

“…This communication pathway has been the subject of active research and is still unresolved and being investigated ( 63 ); it might actually be somewhat different in different polymerase families. A possible suggestion (that could be verified by protein engineering) concerns the ϕVC8 DpoZ insertion 117–127, which corresponds to the region 102–122 in T7 PolA, assumed to be implicated in DNA transfer between pol and exo sites ( 47 ). Additionally, this insertion is in contact with a loop containing the 356-TGR-358 motif, sometimes called pre-Motif A.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Czernecki

Romoli

et al. 2021

Nucleic Acids Research

View full text Add to dashboard Cite

All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3′-5′ exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Czernecki

Romoli

et al. 2021

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…The β-hairpin loop on gp5 may have multiple functions. For instance, a recent study suggests that the β-hairpin loop is important for coordinating polymerase and exonuclease activity [34]. We therefore speculate that the β-hairpin loop might help hold the DNA template in place during exonuclease proofreading.…”

Section: Discussionmentioning

confidence: 83%

“…Moreover, the gp5 polymerase encompasses a positively charged cleft with three lysine residues holding the parental duplex (Figure 1A,B). Previous studies suggested that the β-hairpin loop of gp5 possibly stabilizes the template during DNA synthesis and exonuclease proofreading [34]. However, the role of these two elements during coupled helicase-polymerase replication has not been determined.…”

Section: Introductionmentioning

confidence: 99%

DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication

Gao

2022

IJMS

View full text Add to dashboard Cite

DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular basis of the helicase-polymerase coupling is not fully understood. The recently elucidated T7 replisome structure suggests that the helicase and polymerase sandwich parental DNA and each enzyme pulls a daughter strand in opposite directions. Interestingly, the T7 polymerase, but not the helicase, carries the parental DNA with a positively charged cleft and stacks at the fork opening using a β-hairpin loop. Here, we created and characterized T7 polymerases each with a perturbed β-hairpin loop and positively charged cleft. Mutations on both structural elements significantly reduced the strand-displacement synthesis by T7 polymerase but had only a minor effect on DNA synthesis performed against a linear DNA substrate. Moreover, the aforementioned mutations eliminated synergistic helicase-polymerase binding and unwinding at the DNA fork and processive fork progressions. Thus, our data suggested that T7 polymerase plays a dominant role in helicase-polymerase coupling and replisome progression.

show abstract

“…The DNA template base for the nascent DNA synthesis is 1 nt away Hel and Pol are both motor proteins that can translocate along their DNA substrates. dNTP binding induces the closure of the T7 Pol active site by a finger domain [16,35]. Pyrophosphate release following dNMP addition drives the opening of the finger domain and the translocation of DNA by one nucleotide.…”

Section: Hel-pol Coupling In Bacteriophage T7 Dna Replicationmentioning

confidence: 99%

DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Gao

2021

Viruses

View full text Add to dashboard Cite

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

show abstract

Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site

Cited by 9 publications

References 52 publications

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication

DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Contact Info

Product

Resources

About