Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

García‐Medel, Paola L.; Peralta-Castro, Antolín; Baruch‐Torres, Noe; Fuentes-Pascacio, Alma; Pedroza-García, José Antonio; Cruz‐Ramírez, Alfredo; Brieba, Luis G.

doi:10.1038/s41598-021-00151-7

Cited by 5 publications

(5 citation statements)

References 72 publications

(120 reference statements)

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“…Altogether, research suggests that PrimPol is a highly relevant enzyme in human cells. To note, this enzyme is highly conserved across organisms and species such as: Mus musculus (mice) [ 12 , 13 ], Gallus gallus (chicken) [ 133 ], the plant Arabidopsis thaliana [ 134 ], the archaea Pyrococcus furiosus [ 135 , 136 ] and the bacteria Thermus thermophilus [ 137 , 138 ], highlighting the relevance of this enzyme. Nevertheless, there are organisms such as Saccharomyces cerevisiae (yeast) that do not have PrimPol, in which the PrimPol action is carried out by other enzymes or complexes [ 87 ].…”

Section: Expression Of Human Primpol In Different Tissuesmentioning

confidence: 99%

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

et al. 2022

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DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with convectional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a translesion synthesis (TLS) DNA primase, elongating primers that PrimPol itself sythesizes, or as TLS DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only uses its TLS abilities to facilitate priming across lesions in cells, thus acting as a TLS DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.

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Section: Expression Of Human Primpol In Different Tissuesmentioning

confidence: 99%

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

et al. 2022

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“…Interestingly, plants harbor another primase dubbed PrimPol [ 42 , 43 ]. In Arabidopsis, AtPrimPol is found in the nucleus, mitochondria, and chloroplast.…”

Section: Discussionmentioning

confidence: 99%

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain

Peralta-Castro,

Cordoba-Andrade,

Díaz-Quezada

et al. 2023

BMC Plant Biol

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Background The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). Results Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. Conclusions We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

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“…never exposed to a mutant background) into the resulting heterozygous F1s. The presence of one WT allele in the F1 heterozygotes should be sufficient for WT-like organelle genome maintenance since the mutant alleles of the repair genes of interest are thought to act recessively ( Shedge et al 2007 ; Cappadocia et al 2010 ; Rowan et al 2010 ; Zampini et al 2015 ; Wu et al 2020 ; García-Medel et al 2021 ; Chevigny et al 2022 ). The heterozygous F1s were then allowed to self-cross and we identified three homozygous mutant and three homozygous WT F2s, which were also allowed to self-cross.…”

Section: Methodsmentioning

confidence: 99%

“…2015). OSB2 is a plastid-targeted single-stranded DNA binding protein that has been shown to hamper microhomology-mediated end joining in vitro (García-Medel et al . 2021).…”

Section: Introductionmentioning

confidence: 99%

“…RECA1 is a plastid-targeted protein that has been proposed to act synergistically with plastid whirly proteins to promote plastid genome integrity either by facilitating polymerase lesion bypass or by reversing stalled replication forks ( Rowan et al 2010 ; Zampini et al 2015 ). OSB2 is a plastid-targeted single-stranded DNA binding protein that has been shown to hamper microhomology-mediated end joining in vitro ( García-Medel et al 2021 ). Given that we previously saw a weak signal of increased mtDNA mutation in recA3 mutants ( Wu et al 2020 ), we included another recA3 mutant allele in this study.…”

Section: Introductionmentioning

confidence: 99%

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Disruption of recombination machinery alters the mutational landscape in plant organellar genomes

Waneka,

Broz,

Wold-McGimsey

et al. 2024

Preprint

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Land plant organellar genomes have extremely low rates of point mutation yet also experience high rates of recombination and genome instability. Characterizing the molecular machinery responsible for these patterns is critical for understanding the evolution of these genomes. While much progress has been made towards understanding recombination activity in land plant organellar genomes, the relationship between recombination pathways and point mutation rates remains uncertain. The organellar targetedmutShomolog MSH1 has previously been shown to suppress point mutations as well as non-allelic recombination between short repeats inArabidopsis thaliana. We therefore implemented high-fidelity Duplex Sequencing to test if other genes that function in recombination and maintenance of genome stability also affect point mutation rates. We found small to moderate increases in the frequency of single nucleotide variants (SNVs) and indels in mitochondrial and/or plastid genomes ofA. thalianamutant lines lackingradA,recA1, orrecA3. In contrast,osb2andwhy2mutants did not exhibit an increase in point mutations compared to wild type (WT) controls. In addition, we analyzed the distribution of SNVs in previously generated Duplex Sequencing data fromA. thalianaorganellar genomes and found unexpected strand asymmetries and large effects of flanking nucleotides on mutation rates in WT plants andmsh1mutants. Finally, using long- read Oxford Nanopore sequencing, we characterized structural variants in organellar genomes of the mutant lines and show that different short repeat sequences become recombinationally active in different mutant backgrounds. Together, these complementary sequencing approaches shed light on how recombination may impact the extraordinarily low point mutation rates in plant organellar genomes.

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Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

Cited by 5 publications

References 72 publications

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain

Disruption of recombination machinery alters the mutational landscape in plant organellar genomes

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