Keen BA et al. (2014), Molecular dissection of the domain architecture...

XB-ART-49808

Nucleic Acids Res 2014 May 01;429:5830-45. doi: 10.1093/nar/gku214.

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Molecular dissection of the domain architecture and catalytic activities of human PrimPol.

Keen BA, Jozwiakowski SK, Bailey LJ, Bianchi J, Doherty AJ.

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PrimPol is a primase-polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol-/- cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.

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Species referenced: Xenopus
Genes referenced: cpd primpol

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	Figure 1. Domain architecture of eukaryotic PrimPol. (A) PrimPol is composed of an AEP primase–polymerase and a Zn2+ finger (Zfn) domain. The AEP domain contains three conserved catalytic motifs (I–III). The first motif (motif I, the first two red stars) is the DxE motif that, along with the conserved D in the third motif (motif III, third red star), forms the catalytic triad that coordinates divalent metal ions essential in the synthesis of oligonucleotide chains. The second motif (motif II, two light-orange stars) is a conserved SxH motif that is required for the coordination of the incoming nucleotide. The zinc finger domain contains a canonical C–H–C–C motif that coordinates a zinc ion and stabilises the anti-parallel β-sheet and α helix structure. The numbers below the conserved residues denote their positions in human PrimPol. (B) A number of human PrimPol constructs were produced to analyse the domain architecture of PrimPol. A Zfn knockout mutant was produced by mutating the first conserved cysteine and histidine residues that coordinate the zinc ion (C419A and H426A, respectively). We constructed a number of truncation mutants based on secondary structure predictions. PrimPol1–487 lacks the C-terminus region downstream of the Zfn domain. PrimPol1–354 deletion mutant contains the polymerase domain but lacks the Zfn domain. PrimPol372–560 possesses the zinc finger domain but lacks the polymerase domain. PrimPol1–354/ZF-KO contains only the zinc finger domain but the C419A and H426A are mutated. A 1–334 deletion mutant in Xenopus tropicalis (XPrimPol1–334) was additionally constructed that is equivalent to PrimPol1–354.
	Figure 2. Primase activity of human PrimPol. (A) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. (B) PrimPolZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. (C) PrimPol1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. (D) PrimPol1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
	Figure 3. Polymerase activity and fidelity of human PrimPol. (A) Human PrimPol was incubated with dNTPs and substrate at 1, 3, 5 and 30 min time points. PrimPol was proficient at extending an undamaged oligonucleotide template using dNTPs. Human PrimPol did not require an intact zinc finger in order to carry out primer extension, as evidenced by the extension of primers by PrimPolZF-KO and PrimPol1–354. PrimPol1–487 that lacked the unstructured C-terminus of the protein was also polymerase proficient. PrimPol1–354 exhibited a higher rate of polymerase activity compared to the other constructs. (B) Incorporation of nucleotides opposite two templating cytosine bases. PrimPol was incubated for 5 min with the DNA substrate and each of the dNTPs. All four of these PrimPol constructs inserted two guanine nucleotides opposite two cytosines in Watson–Crick base-pairing manner. PrimPol1–354 could additionally incorporate a single adenine opposite the first cytosine.
	Figure 4. Template-independent extension in the presence of manganese. Human PrimPol was incubated for 30 min with DNA substrate and each of the dNTPs in the presence of manganese. (A) Wild-type PrimPol was unable to extend from a ds DNA template with a blunt end, but could extend from a primer annealed to an overhanging template, even synthesising long tracts of homopolymers. (B) PrimPolZF-KO could extend from an overhanging DNA template in the presence of manganese and, consistent with the wild-type, did not extend from a ds DNA substrate. The incorporation of 1 or 2 nucleotides of guanine or adenine opposite an overhanging template suggests that PrimPolZF-KO incorporates in a low-fidelity template-dependent manner when incubated with overhanging DNA. (C) PrimPol1–487 exhibited a highly similar terminal transferase activity spectrum to the PrimPolZF-KO. In the presence of manganese, it incorporated bases opposite an overhang in a low fidelity, template-dependent manner. (D) PrimPol1–354 also exhibited low fidelity extension of a primer annealed to an overhanging template in the presence of manganese.
	Figure 5. PrimPol's polymerase domain binds ss and ds DNA but the zinc finger domain binds only ss DNA. (A) PrimPol1–354 was tested for its ability to bind DNA by shift assays. PrimPol polymerase domain was incubated with 40 nM DNA at various protein concentrations (0.05, 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 μM) for 60 min at 25°C. The polymerase domain binds to ds DNA and ss DNA with approximately equal proficiency. Binding was evident at concentrations ∼0.5 μM but a complete shift was observed at 5.0 μM for each DNA substrate tested. (B) PrimPol372–560 (Zfn domain) was also assayed for binding to DNA activity. It was incubated with 40 nM DNA at various protein concentrations (0.05, 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 μM). A DNA shift was not observed with ds DNA substrate but a clear shift was evident with ss DNA, suggesting that the zinc finger binds single-stranded regions of DNA. (C) PrimPol372–560/ZF-KO, lacking a functional Zfn, was incubated with 40 nM DNA at the same protein concentrations and ss DNA binding activity was not observed.
	Figure 6. PrimPol1–354 can replicate through CPD and (6–4(PP)) lesions. (A) PrimPol1–354 was incubated with a primer-template substrate in which the template contained a CPD lesion downstream of the primer-template junction in the presence of dNTPs. Time points were taken between 0.5 and 60 min. PrimPol1–354 extends from the primer up to the CPD, before stalling, it will then added a base opposite the first thymine of the CPD and continue to extend until the end of the template (left panel). PrimPol1–354 was then incubated with each dNTP to test which nucleotide it incorporates opposite a CPD (right panels). PrimPol1–354 incorporated adenine opposite the first and second thymine of the CPD. (B) The polymerase domain of PrimPol can also perform TLS bypass of a 6–4(PP) lesion immediately downstream of the primer-template junction (left panel). PrimPol1–354 incorporates either an adenine or cytosine nucleotide opposite the first thymine of the 6–4(PP) (right panels). If an adenine is incorporated opposite the first thymine, a cytosine or thymine is then incorporated opposite the second. If a cytosine is incorporated opposite the first thymine, an adenine, cytosine or thymine will be incorporated opposite the second thymine of the 6–4(PP).
	Figure 7. PrimPol1–354 can replicate through an 8-oxoguanine (8oxoG) lesion and a deoxyuracil (dU) base. (A) The polymerase domain exhibited minimal stalling opposite an 8oxoG lesion and extended fully to the end of the template. PrimPol incorporated an adenine or cytosine opposite the 8oxoG, which are the expected bases to be incorporated by Hoogsteen base pairing or Watson–Crick base pairing, respectively. (B) PrimPol1–354 efficiently synthesised through a templating dU site. PrimPol incorporates an adenine nucleotide opposite the dU lesion through Watson–Crick base pairing.
	Figure 8. Processivity of the polymerase activity of PrimPol. PrimPol was pre-incubated for 30 min at 37°C with an undamaged DNA primer-template substrate to allow PrimPol to bind to the DNA. The reaction was initiated by the addition of dNTPs and an excess of sonicated herring sperm DNA (trap) and time points taken at 15, 30, 60, 120 and 360 s. (A) After 360 s, wild-type PrimPol incorporated up to 4 nucleotides opposite the template but a significant fraction of enzyme incorporated only 1, 2 or 3 nucleotides (left panel). To confirm that the trap prevents polymerase extending from a second template, the trap was also added into the pre-incubation mix with PrimPol and the DNA substrate. This reaction was supplemented with dNTPs and there is no extension (right panel), thus successfully exhibiting the effectiveness of the trap. (B) PrimPol1–354 also predominantly incorporates up to 4 nucleotides but there were fewer polymerases incorporating only 1, 2 or 3 nucleotides (left panel). The effectiveness of the trap was also successfully confirmed (right panel). (C) Percentage of PrimPol molecules incorporating at least n dNTPs for either full-length PrimPol or PrimPol1–354, calculated using Equation (1).
	Figure 9. PrimPol's zinc finger domain is required for UV lesion bypass in vivo. (A) PrimPol−/− DT40 cells were complemented with variants of PrimPol and their in vivo expression confirmed by western blot. (B) Unperturbed replication fork rates were analysed by DNA fibre analysis using CldU and IdU labels for all the complemented cell types. The data represent the mean of three or more experiments with error bars showing the mean standard deviation. (C) DNA fork rate analysis was also carried out with after a 20 J/m2 pulse of UV, between CldU and IdU labels, to look for fork-stalling. Data is shown as the ratio of the two labels, Cldu, pre-UV : IdU, post UV label (n = 3). The black lines indicate the Cldu:IdU ratio of the two labels in the wild-type cells and the red lines indicate the ratios in cells complemented with the indicated PrimPol variants.
	Figure 10. Models of the mechanisms of action of PrimPol. (A) PrimPol has both DNA primase and polymerase activities. The zinc finger of PrimPol stabilises the binding of the polymerase domain on single-stranded DNA. The AEP polymerase domain coordinates two adjacent deoxynucleotides opposite the single-stranded DNA template and the stability afforded to this complex by the Zfn binding allows synthesis of a dinucleotide. This dinucleotide can then be extended in a more processive way to form longer products. PrimPol can also bind primer-template junctions to catalyse more canonical primer extension reactions. Both the polymerase and primase activities are intrinsic to the AEP domain. Although the zinc finger is essential for maintaining less ‘stable' primer synthesis, it is also important for modulating the fidelity and processivity of the polymerase activities. This stabilizing influence of the Zfn may be particularly important for allowing productive synthesis during TLS bypass of distorting lesions and structures. We postulate that the primase and polymerase activities of PrimPol are not discrete activities but rather represent different synthesis modes performed under different DNA binding conditions. (B) PrimPol uses these activities to bypass DNA damage that blocks replicative polymerases. When replicative polymerases encounter a blocking lesion, they are displaced or regress from the site of damage. PrimPol can then access the template upstream of the lesion, where it can reprime following the lesion (left) or if it can access the primer-template junction it can carry out TLS (right). Once PrimPol-mediated bypass of the lesion has occurred, the replicative polymerase can then resume replication at the fork.

References [+] :

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