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<StructureSection load='7pbk' size='340' side='right' caption='Vibriophage ΦVC8 DNA polymerase DpoZ deposited under the PDB ID [https://www.rcsb.org/structure/7PBK 7pbk].' scene='90/909993/Open_domains_colored/1'>
<StructureSection load='7pbk' size='340' side='right' caption='Vibriophage ΦVC8 DNA polymerase DpoZ deposited under the PDB ID [https://www.rcsb.org/structure/7PBK 7pbk]; thumb-exo open conformation.' scene='90/909993/Open_domains_colored/1'>
== Introduction ==
== Introduction ==
The vibriophage ΦVC8 DNA polymerase DpoZ is a [[DNA polymerase]] belonging to the PolA family and the ΦVC8-like DpoZ subfamily, a group currently identified in certain species of bacteriophages. DpoZ consists of two subfamilies: ΦVC8-like and Wayne-like. These polymerases confer selectivity in addition of the nucleobase 2-aminoadenine (Z) over adenine (A), with A completely ablated from their genomes. Z forms a non Watson-Crick base pair with thymine (T) consisting of three hydrogen bonds as opposed to the two present in A-T base pairing. Z is a relatively novel discovery, having only recently had its biosynthetic pathway described in detail. DNA modifications in bacteriophages usually confer selective advantages by allowing phages to avoid host cell restriction enzyme digestion of their genomes. The phage S-2L, which encodes a PrimPol polymerase, contains a Z-specific analog of the purine nucleotide enzyme PurA (link) known as PurZ. Polymerases specific to Z are required to incorporate the nucleotide completely over A into phage genomes, and as noted include DpoZ polymerases as well as the as-yet uncharacterized PrimPol identified in phage S-2L. The mechanisms by which these polymerases carry out these functions are still under investigation, though specific structural feature and putative specificity mechanisms are highlighted below.
[[Vibriophage phiVC8 DpoZ]] is a [[DNA polymerase]] belonging to the PolA family and the ΦVC8-like DpoZ subfamily, a group currently identified in certain species of bacteriophages<ref>PMID:34751404</ref>. DpoZ consists of two subfamilies: ΦVC8-like and Wayne-like. These polymerases confer selectivity in addition of the nucleobase 2-aminoadenine (Z) over adenine (A), with A completely ablated from their genomes. Z forms a non Watson-Crick base pair with thymine (T) consisting of three hydrogen bonds as opposed to the two present in A-T base pairing. Z is a relatively novel discovery, having only recently had its biosynthetic pathway described in detail<ref>PMID:33926954</ref>. DNA modifications in bacteriophages usually confer selective advantages by allowing phages to avoid host cell restriction enzyme digestion of their genomes<ref>Weigele, P., & Raleigh, E. A. (2016). Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses. <i>Chemical Reviews</i>, <i>116</i>(20), 12655–12687. https://doi.org/10.1021/acs.chemrev.6b00114</ref>. The phage S-2L has a Z genome and encodes a [https://www.ncbi.nlm.nih.gov/gene/201973 PrimPol polymerase] as well as a nucleotide phosphohydrolase called [https://www.rcsb.org/structure/6ZPB DatZ] that removes the triphosphate tail of only dATP, excluding it from the genome<ref>PMID:33893297</ref>. Vibriophage ΦVC8 also encodes DatZ<ref>PMID:34751404</ref>, though it is unknown if it is the primary enzyme responsible for A exclusion or if its polymerase also plays a role. The specific mechanisms by which ΦVC8-like DpoZ polymerases carry out A exclusion and Z inclusion are still under investigation, though specific structural features and putative specificity mechanisms are highlighted below.


== Structure ==
== Structural Highlights ==
The 2.8A crystal structure solved of the 646 amino acid DpoZ by Czernecki et al contains two domains: a <scene name='90/909993/Polymerase_domain/5'>polymerase domain</scene> and a  
The 2.8Å crystal structure solved of the 646 amino acid protein contains two domains: a <scene name='90/909993/Polymerase_domain/7'>polymerase domain</scene> and a  
<scene name='90/909993/Exonuclease_domain/2'>3'-5' exonuclease domain</scene>. ΦVC8 DpoZ closely resembles <i>E. coli</i> [[DNA Polymerase I]] Klenow fragment, containing distinct palm, thumb, and fingers subdomains in addition to the exonuclease domain. The enzyme exhibits the typical fold of PolA polymerases including <i>E. coli</i> [https://www.rcsb.org/structure/1KFD Klenow fragment] and [https://www.rcsb.org/structure/1T7P T7 DNA polymerase]. The palm subdomain contains the polymerase active site, where the thumb and fingers clamp onto a DNA substrate to hold it in place.  
<scene name='90/909993/Exonuclease_domain/4'>3'-5' exonuclease domain</scene><ref>PMID:34751404</ref>. [[Image:Openclosed 7pbk.png|300px|left|thumb| Open and closed conformation overlay. Open in <font color=pink>pink</font>, closed in <font color=cyan>cyan</font>.]] The structure deposited in PDB (7pbk) has two conformations: <scene name='90/909993/Exo_open/5'>thumb-exo open</scene> and <scene name='90/909993/Exo_closed/3'>thumb-exo closed</scene>. These conformations involve movement of the thumb and exonuclease domains. ΦVC8 DpoZ closely resembles <i>E. coli</i> [[DNA Polymerase I]] Klenow fragment, containing distinct <scene name='90/909993/Pol_subdomains/1'>palm, thumb, and fingers subdomains</scene> (palm in <font color='blue'>'''blue'''</font>, thumb in <font color='green'>'''green'''</font>, fingers in <font color='pink'>'''pink'''</font>, subdomains approximated from related polymerases). The enzyme exhibits the typical fold of PolA polymerases including <i>E. coli</i> [https://www.rcsb.org/structure/1KFD Klenow fragment] and [https://www.rcsb.org/structure/1T7P T7 DNA polymerase]. The residues <scene name='90/909993/K162g276/1'>K162 and G276</scene> appear to have the largest positional shifts between the two conformations. The palm subdomain contains the <scene name='90/909993/Polymerase_active_site/1'>polymerase active site</scene>, where the thumb and fingers clamp onto a DNA substrate to hold it in place. The 3'-5' exonuclease site has a shift in residues R161 and M165 from the <scene name='90/909993/Exonuclease_active_open/1'>open conformation</scene> and the <scene name='90/909993/Exo_activesite_closed/1'>closed conformation</scene>. [[Image:R161m165 overlay.png|300px|left|thumb| Comparison of Arg161 and Met165 residues in <font color=magenta>open</font> and <font color=cyan>closed</font> conformations.]]
The structure from Czernecki et al contains two conformations: <scene name='90/909993/Exo_open/2'>thumb-exo open</scene> and <scene name='90/909993/Exo_closed/1'>thumb-exo closed</scene>. These conformations involve movement of the thumb and exonuclease domains, and the closed conformation excludes binding of DNA when modeled with dsDNA. The residues K162 and G276 appear to have the largest positional shifts between the two conformations.


When aligned with other PolA polymerases, three unique insertions in the polymerase domain are present. The first ranges from the residues 349-353 (scene) in the palm subdomain. The second is in helix O in the fingers subdomain from residues 442-448 (scene). The third is between helices O1 and P on the tip of the fingers, from residues 473-491 (scene). The longest of these insertions is very flexible, not being well defined on the electron density map (scene?) and indicating that DNA binding may be necessary for stabilization of that loop. The first and third insertions likely interact with dsDNA, though solving a ternary complex structure is required to confirm this. The second insertion (residues 442-448 (scene)) adds a loop in the helix structure that normally interacts with dNTPs.  
When aligned with other prokaryotic PolA polymerases<ref>PMID:34751404</ref>, three unique insertions in the polymerase domain are present. The first ranges from the residues <scene name='90/909993/Insertion1/2'>349-353</scene> in the palm subdomain. The second is adjacent to <scene name='90/909993/Insertion_2/1'>helix O in the fingers subdomain from residues 442-448</scene>, with this insertion disrupting a part of motif B containing helix O. The third is between helices O1 and P on the tip of the fingers, from <scene name='90/909993/Insertion_3/1'>residues 473-491</scene>. The longest of these insertions (3) is very flexible, not being well defined on the electron density map, indicating that DNA binding may be necessary for stabilization of that loop. The first and third insertions likely interact with dsDNA, though solving a ternary complex structure is required to confirm this. The second insertion (residues 442-448) adds a <scene name='90/909993/Insertion_2/2'>loop in the helix structure</scene> that normally interacts with dNTPs<ref>Miller, B.R., Beese,L.S., Parish, C.A. and Wu,E.Y. (2015) The closing mechanism of DNA polymerase I at atomic resolution. <i>Structure</i>, <i>23</i>,1609–1620. https://doi.org/10.1016/j.str.2015.06.016</ref>.


The exonuclease domain is larger and contains noncanonical alpha helices compared to other members of the PolA family. These helices are referred to as E1 and E2 (scene). This domain also contains an insertion between helices alpha4 and alpha5 (scene) present in other phages such as T7 and implicated in shuttling the 3' end of the DNA primer between the polymerase and exonuclease sites. Some critical residues for catalysis such as a universally conserved HD catalytic dyad (scene) are present in ΦVC8 DpoZ, as these are required for polymerase activity. Some crucial residues are lacking in the structure, which may result in loss or alteration of some proofreading activity of the enzyme. However, the critical arginine residue responsible for stabilizing the gamma phosphate of incoming dNTPs is present at position 440 (scene). The enzyme also includes point mutations present in other DpoZ including L455, F459, and G548 (scene). These residues are present near or in the polymerase domain active site, and residues corresponding to these exist in PolA polymerases that do not incorporate Z. L455 and G548 (scene) do not appear in any known Wayne-like DpoZ subfamily structures, though the F459 (scene) residue is present. F459 is normally a tyrosine residue that acts as a steric gate for distinguishing dNTPs from NTPs(6), though this function may be conserved as was shown for T7 phage with a Tyr-->Phe mutation. In any case, these individual changes from other polymerases may help account for specificity in Z nucleobase recognition, as it is likely that it is not a single mutation in the enzyme that accounts for the selectivity.
== Function ==
===Polymerase domain===
The <scene name='90/909993/Polymerase_active_site/1'>polymerase active site</scene> contains 10 residues essential to catalyzing nucleotide addition to the template strand. The universally conserved
<scene name='90/909993/Polymerase_active_site_dyad/1'>catalytic dyad (H581 and D582)</scene> is present and key residue R440, responsible for stabilizing the gamma phosphate of incoming dNTPs, is also conserved. Still, a number of residues in the polymerase domain differ from other PolA polymerases. <scene name='90/909993/Polymerase_dpoz_residues/1'>L455, F459, G548, and S583</scene> are all conserved mutations in the ΦVC8 DpoZ subfamily, though not in the Wayne-like DpoZ subfamily<ref>PMID:34751404</ref>. L455 and G548 do not appear in any known Wayne-like DpoZ subfamily structures, though the F459 residue is present. F459 is normally a tyrosine residue that acts as a steric gate for distinguishing dNTPs from NTPs<ref>Tabor, S., & Richardson, C. C. (1995). A single residue in DNA polymerases of the <i>Escherichia coli</i> DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. <i>Proceedings of the National Academy of Sciences of the United States of America</i>, <i>92</i>(14), 6339–6343. https://doi.org/10.1073/pnas.92.14.6339</ref>, though this function may be conserved as was shown for <i>T. aquaticus</i> DNA polymerase with a Tyr to Phe mutation in a previous study<ref>Suzuki, M., Baskin, D., Hood, L., & Loeb, L. A. (1996). Random mutagenesis of <i>Thermus aquaticus</i> DNA polymerase I: concordance of immutable sites in vivo with the crystal structure. <i>Proceedings of the National Academy of Sciences of the United States of America</i>, <i>93</i>(18), 9670–9675. https://doi.org/10.1073/pnas.93.18.9670</ref>. In any case, these individual changes from other polymerases may help account for specificity in Z nucleobase recognition, as it is likely that it is not a single mutation in the enzyme that accounts for the selectivity.


== Function ==
===Exonuclease domain===
The primary unique feature of ΦVC8 DpoZ and other DpoZ family members is its selectivity in Z vs A incorporation. The exact mechanism by which this occurs is still under investigation and cannot be definitively described, though there is a proposed mechanism based off of the currently solved structure. The noncanonical helices E1 and E2 (scene) comprise an unusually mobile portion of the exonuclease domain that may contribute to proofreading properties. Residues R161 and M165 of E2 (scene) have been shown to contact the base of leaving nucleotides when modeled. Mutating these residues in a double mutation to alanine resulted in a detrimental effect in exonuclease activity and about equivalent efficiencies of Z and A incorporation, though interestingly also resulted in a higher incorporation of Z over A that has not been investigated or accounted for. These residues may still have an important function in Z vs A incorporation that requires further investigation.  
The exonuclease domain of ΦVC8 DpoZ is larger and contains two non-canonical helices when compared with other PolA family members. These helices are referred to as <scene name='90/909993/Helix_e1/1'>E1</scene> and  
<scene name='90/909993/Helix_e2/1'>E2</scene>. This domain also contains an <scene name='90/909993/Exo_insertion/2'>insertion</scene> between helices a4 and a5 present in other phages, such as T7, and is implicated in shuttling the 3' end of the DNA primer between the polymerase and exonuclease sites<ref>Juarez-Quintero, V., Peralta-Castro, A., Benítez Cardoza, C. G., Ellenberger, T. & Brieba, L. G. (2021). Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site. <i>Biochemical Journal</i>, <i>478</i>, 2665–2679https://doi.org/10.1042/BCJ20200922</ref>.


Czernecki et al propose that selectivity of Z vs A may result from the enzyme exhibiting selectivity in incorporating base pairs with three hydrogen bonds and excluding those with two (A-T). This could occur by polymerase backtracking, a feature previously thought to be distinct to RNA-dependent DNA polymerases but has also been shown to occur in some DNA-dependent DNA polymerases. Polymerase backtracking occurs when the enzyme disengages from the nascent 3' end of DNA and moves backwards on the template strand. This proposed mechanism may allow the enzyme to be more sensitive to base-pairing interactions and differentiate between two vs three hydrogen bonds in a base pair. This ability would be enhanced in the enzyme compared to other polymerases and the energy barrier between backtracking conformation and forward moving polymerization would need be to significantly lowered. This may work through an allosteric mechanism that gradually allows the transition of the enzyme from the polymerase domain to the exonuclease domain to excise any bases with fewer than three hydrogen bonds. The insertion at residues 117-127 (scene) may be involved in such a mechanism, though this is yet to be tested and would require analysis of a ternary complex. Further investigation into the mechanisms behind Z incorporation and selectivity is ongoing and may soon shed more light on the specific structural feature necessary to facilitate such specificity.
The primary feature of ΦVC8 DpoZ and other DpoZ family members is its selectivity in Z vs A incorporation. The exact mechanism by which this occurs is still under investigation and cannot be definitively described, though there is a proposed mechanism based off of the currently solved structure ([https://www.rcsb.org/structure/7PBK 7pbk]). The noncanonical helices E1 and E2 comprise an unusually mobile portion of the exonuclease domain that may contribute to proofreading properties. Residues <scene name='90/909993/Helix_e2_r161m165/2'>R161 and M165</scene> of helix E2 have been shown to contact the base of leaving nucleotides when modeled. Mutating these residues in a double mutation to alanine resulted in a detrimental effect in exonuclease activity and about equivalent efficiencies of Z and A incorporation, though interestingly also resulted in a higher incorporation of Z over A that has not been investigated or accounted for. These residues may still have an important function in Z vs A incorporation that requires further investigation.


===2-aminoadenine Incorporation===
Selectivity of Z vs A may result from the enzyme exhibiting selectivity in incorporating base pairs with three hydrogen bonds and excluding those with two (A-T). This could occur by polymerase backtracking, a feature previously thought to be distinct to RNA-dependent DNA polymerases but has also been shown to occur in some DNA-dependent DNA polymerases<ref>Samson, C., Legrand,P., Tekpinar,M., Rozenski,J., Abramov,M., Holliger,P., Pinheiro,V.B., Herdewijn, P. and Delarue,M. (2020) Structural studies of HNA substrate specificity in mutants of an archaeal DNA polymerase obtained by directed evolution. <i>Biomolecules</i>, <i>10</i>, 1647.</ref>. Polymerase backtracking occurs when the enzyme disengages from the nascent 3' end of DNA and moves backwards on the template strand. This proposed mechanism may allow the enzyme to be more sensitive to base-pairing interactions and differentiate between two vs three hydrogen bonds in a base pair. This ability would be enhanced in the enzyme compared to other polymerases and the energy barrier between backtracking conformation and forward moving polymerization would need be to significantly lowered. This may work through an allosteric mechanism that gradually allows the transition of the enzyme from the polymerase domain to the exonuclease domain to excise any bases with fewer than three hydrogen bonds. The insertion at residues <scene name='90/909993/Exo_insertion/2'>117-127</scene> of the exonuclease domain may be involved in such a mechanism, though this is yet to be tested and would require analysis of a ternary complex. Further investigation into the mechanisms behind Z incorporation and selectivity is ongoing and may soon shed more light on the specific structural feature necessary to facilitate such specificity.
</StructureSection>
</StructureSection>
== References ==
== References ==
1. Czernecki, D., Hu, H., Romoli, F., & Delarue, M. (2021). Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage VC8. <i>Nucleic Acids Research</i>, <i>49</i>(20), 11974–11985. https://doi.org/10.1093/nar/gkab955
2. Zhou, Y., Xu, X., Wei, Y., Cheng, Y., Guo, Y., Khudyakov, I., Liu, F., He, P., Song, Z., Li, Z., Gao, Y., Ang, E. L., Zhao, H., Zhang, Y., & Zhao, S. (2021). A widespread pathway for substitution of adenine by diaminopurine in phage genomes. <i>Science</i>, <i>372</i>(6541), 512–516. https://doi.org/10.1126/science.abe4882
3. Weigele, P., & Raleigh, E. A. (2016). Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses. <i>Chemical Reviews</i>, <i>116</i>(20), 12655–12687. https://doi.org/10.1021/acs.chemrev.6b00114
4. Miller, B.R., Beese,L.S., Parish, C.A. and Wu,E.Y. (2015) The closing mechanism of DNA polymerase I at atomic resolution. <i>Structure</i>, <i>23</i>,1609–1620. https://doi.org/10.1016/j.str.2015.06.016
5. Juarez-Quintero, V., Peralta-Castro, A., Benítez Cardoza, C. G., Ellenberger, T. & Brieba, L. G. (2021). Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site. <i>Biochemical Journal</i>, <i>478</i>, 2665–2679https://doi.org/10.1042/BCJ20200922
6. Tabor, S., & Richardson, C. C. (1995). A single residue in DNA polymerases of the <i>Escherichia coli</i> DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. <i>Proceedings of the National Academy of Sciences of the United States of America</i>, <i>92</i>(14), 6339–6343. https://doi.org/10.1073/pnas.92.14.6339
7. Suzuki, M., Baskin, D., Hood, L., & Loeb, L. A. (1996). Random mutagenesis of <i>Thermus aquaticus</i> DNA polymerase I: concordance of immutable sites in vivo with the crystal structure. <i>Proceedings of the National Academy of Sciences of the United States of America</i>, <i>93</i>(18), 9670–9675. https://doi.org/10.1073/pnas.93.18.9670
8. Samson, C., Legrand,P., Tekpinar,M., Rozenski,J., Abramov,M., Holliger,P., Pinheiro,V.B., Herdewijn, P. and Delarue,M. (2020) Structural studies of HNA substrate specificity in mutants of an archaeal DNA polymerase obtained by directed evolution. <i>Biomolecules</i>, <i>10</i>, 1647.
<references/>
<references/>

Latest revision as of 16:21, 3 May 2022

Introduction

Vibriophage phiVC8 DpoZ is a DNA polymerase belonging to the PolA family and the ΦVC8-like DpoZ subfamily, a group currently identified in certain species of bacteriophages[1]. DpoZ consists of two subfamilies: ΦVC8-like and Wayne-like. These polymerases confer selectivity in addition of the nucleobase 2-aminoadenine (Z) over adenine (A), with A completely ablated from their genomes. Z forms a non Watson-Crick base pair with thymine (T) consisting of three hydrogen bonds as opposed to the two present in A-T base pairing. Z is a relatively novel discovery, having only recently had its biosynthetic pathway described in detail[2]. DNA modifications in bacteriophages usually confer selective advantages by allowing phages to avoid host cell restriction enzyme digestion of their genomes[3]. The phage S-2L has a Z genome and encodes a PrimPol polymerase as well as a nucleotide phosphohydrolase called DatZ that removes the triphosphate tail of only dATP, excluding it from the genome[4]. Vibriophage ΦVC8 also encodes DatZ[5], though it is unknown if it is the primary enzyme responsible for A exclusion or if its polymerase also plays a role. The specific mechanisms by which ΦVC8-like DpoZ polymerases carry out A exclusion and Z inclusion are still under investigation, though specific structural features and putative specificity mechanisms are highlighted below.

Structural Highlights

The 2.8Å crystal structure solved of the 646 amino acid protein contains two domains: a and a

[6].

Open and closed conformation overlay. Open in pink, closed in cyan.

The structure deposited in PDB (7pbk) has two conformations: and . These conformations involve movement of the thumb and exonuclease domains. ΦVC8 DpoZ closely resembles E. coli DNA Polymerase I Klenow fragment, containing distinct (palm in blue, thumb in green, fingers in pink, subdomains approximated from related polymerases). The enzyme exhibits the typical fold of PolA polymerases including E. coli Klenow fragment and T7 DNA polymerase. The residues appear to have the largest positional shifts between the two conformations. The palm subdomain contains the , where the thumb and fingers clamp onto a DNA substrate to hold it in place. The 3'-5' exonuclease site has a shift in residues R161 and M165 from the and the .

Comparison of Arg161 and Met165 residues in open and closed conformations.

When aligned with other prokaryotic PolA polymerases[7], three unique insertions in the polymerase domain are present. The first ranges from the residues in the palm subdomain. The second is adjacent to , with this insertion disrupting a part of motif B containing helix O. The third is between helices O1 and P on the tip of the fingers, from . The longest of these insertions (3) is very flexible, not being well defined on the electron density map, indicating that DNA binding may be necessary for stabilization of that loop. The first and third insertions likely interact with dsDNA, though solving a ternary complex structure is required to confirm this. The second insertion (residues 442-448) adds a that normally interacts with dNTPs[8].

Function

Polymerase domain

The contains 10 residues essential to catalyzing nucleotide addition to the template strand. The universally conserved

is present and key residue R440, responsible for stabilizing the gamma phosphate of incoming dNTPs, is also conserved. Still, a number of residues in the polymerase domain differ from other PolA polymerases. are all conserved mutations in the ΦVC8 DpoZ subfamily, though not in the Wayne-like DpoZ subfamily[9]. L455 and G548 do not appear in any known Wayne-like DpoZ subfamily structures, though the F459 residue is present. F459 is normally a tyrosine residue that acts as a steric gate for distinguishing dNTPs from NTPs[10], though this function may be conserved as was shown for T. aquaticus DNA polymerase with a Tyr to Phe mutation in a previous study[11]. In any case, these individual changes from other polymerases may help account for specificity in Z nucleobase recognition, as it is likely that it is not a single mutation in the enzyme that accounts for the selectivity.

Exonuclease domain

The exonuclease domain of ΦVC8 DpoZ is larger and contains two non-canonical helices when compared with other PolA family members. These helices are referred to as and

. This domain also contains an between helices a4 and a5 present in other phages, such as T7, and is implicated in shuttling the 3' end of the DNA primer between the polymerase and exonuclease sites[12].

The primary feature of ΦVC8 DpoZ and other DpoZ family members is its selectivity in Z vs A incorporation. The exact mechanism by which this occurs is still under investigation and cannot be definitively described, though there is a proposed mechanism based off of the currently solved structure (7pbk). The noncanonical helices E1 and E2 comprise an unusually mobile portion of the exonuclease domain that may contribute to proofreading properties. Residues of helix E2 have been shown to contact the base of leaving nucleotides when modeled. Mutating these residues in a double mutation to alanine resulted in a detrimental effect in exonuclease activity and about equivalent efficiencies of Z and A incorporation, though interestingly also resulted in a higher incorporation of Z over A that has not been investigated or accounted for. These residues may still have an important function in Z vs A incorporation that requires further investigation.

2-aminoadenine Incorporation

Selectivity of Z vs A may result from the enzyme exhibiting selectivity in incorporating base pairs with three hydrogen bonds and excluding those with two (A-T). This could occur by polymerase backtracking, a feature previously thought to be distinct to RNA-dependent DNA polymerases but has also been shown to occur in some DNA-dependent DNA polymerases[13]. Polymerase backtracking occurs when the enzyme disengages from the nascent 3' end of DNA and moves backwards on the template strand. This proposed mechanism may allow the enzyme to be more sensitive to base-pairing interactions and differentiate between two vs three hydrogen bonds in a base pair. This ability would be enhanced in the enzyme compared to other polymerases and the energy barrier between backtracking conformation and forward moving polymerization would need be to significantly lowered. This may work through an allosteric mechanism that gradually allows the transition of the enzyme from the polymerase domain to the exonuclease domain to excise any bases with fewer than three hydrogen bonds. The insertion at residues of the exonuclease domain may be involved in such a mechanism, though this is yet to be tested and would require analysis of a ternary complex. Further investigation into the mechanisms behind Z incorporation and selectivity is ongoing and may soon shed more light on the specific structural feature necessary to facilitate such specificity.

Vibriophage ΦVC8 DNA polymerase DpoZ deposited under the PDB ID 7pbk; thumb-exo open conformation.

Drag the structure with the mouse to rotate

ReferencesReferences

  1. ↑ Czernecki D, Hu H, Romoli F, Delarue M. Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage varphiVC8. Nucleic Acids Res. 2021 Nov 18;49(20):11974-11985. doi: 10.1093/nar/gkab955. PMID:34751404 doi:http://dx.doi.org/10.1093/nar/gkab955
  2. ↑ Zhou Y, Xu X, Wei Y, Cheng Y, Guo Y, Khudyakov I, Liu F, He P, Song Z, Li Z, Gao Y, Ang EL, Zhao H, Zhang Y, Zhao S. A widespread pathway for substitution of adenine by diaminopurine in phage genomes. Science. 2021 Apr 30;372(6541):512-516. doi: 10.1126/science.abe4882. PMID:33926954 doi:http://dx.doi.org/10.1126/science.abe4882
  3. ↑ Weigele, P., & Raleigh, E. A. (2016). Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses. Chemical Reviews, 116(20), 12655–12687. https://doi.org/10.1021/acs.chemrev.6b00114
  4. ↑ Czernecki D, Legrand P, Tekpinar M, Rosario S, Kaminski PA, Delarue M. How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA. Nat Commun. 2021 Apr 23;12(1):2420. doi: 10.1038/s41467-021-22626-x. PMID:33893297 doi:http://dx.doi.org/10.1038/s41467-021-22626-x
  5. ↑ Czernecki D, Hu H, Romoli F, Delarue M. Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage varphiVC8. Nucleic Acids Res. 2021 Nov 18;49(20):11974-11985. doi: 10.1093/nar/gkab955. PMID:34751404 doi:http://dx.doi.org/10.1093/nar/gkab955
  6. ↑ Czernecki D, Hu H, Romoli F, Delarue M. Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage varphiVC8. Nucleic Acids Res. 2021 Nov 18;49(20):11974-11985. doi: 10.1093/nar/gkab955. PMID:34751404 doi:http://dx.doi.org/10.1093/nar/gkab955
  7. ↑ Czernecki D, Hu H, Romoli F, Delarue M. Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage varphiVC8. Nucleic Acids Res. 2021 Nov 18;49(20):11974-11985. doi: 10.1093/nar/gkab955. PMID:34751404 doi:http://dx.doi.org/10.1093/nar/gkab955
  8. ↑ Miller, B.R., Beese,L.S., Parish, C.A. and Wu,E.Y. (2015) The closing mechanism of DNA polymerase I at atomic resolution. Structure, 23,1609–1620. https://doi.org/10.1016/j.str.2015.06.016
  9. ↑ Czernecki D, Hu H, Romoli F, Delarue M. Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage varphiVC8. Nucleic Acids Res. 2021 Nov 18;49(20):11974-11985. doi: 10.1093/nar/gkab955. PMID:34751404 doi:http://dx.doi.org/10.1093/nar/gkab955
  10. ↑ Tabor, S., & Richardson, C. C. (1995). A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proceedings of the National Academy of Sciences of the United States of America, 92(14), 6339–6343. https://doi.org/10.1073/pnas.92.14.6339
  11. ↑ Suzuki, M., Baskin, D., Hood, L., & Loeb, L. A. (1996). Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure. Proceedings of the National Academy of Sciences of the United States of America, 93(18), 9670–9675. https://doi.org/10.1073/pnas.93.18.9670
  12. ↑ Juarez-Quintero, V., Peralta-Castro, A., Benítez Cardoza, C. G., Ellenberger, T. & Brieba, L. G. (2021). Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site. Biochemical Journal, 478, 2665–2679https://doi.org/10.1042/BCJ20200922
  13. ↑ Samson, C., Legrand,P., Tekpinar,M., Rozenski,J., Abramov,M., Holliger,P., Pinheiro,V.B., Herdewijn, P. and Delarue,M. (2020) Structural studies of HNA substrate specificity in mutants of an archaeal DNA polymerase obtained by directed evolution. Biomolecules, 10, 1647.

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