TY - JOUR
T1 - Biochemical and structural characterization of the first-discovered metazoan DNA cytosine-N4 methyltransferase from the bdelloid rotifer Adineta vaga
AU - Zhou, Jujun
AU - Horton, John R.
AU - Kaur, Gundeep
AU - Chen, Qin
AU - Li, Xuwen
AU - Mendoza, Fabian
AU - Wu, Tao
AU - Blumenthal, Robert M.
AU - Zhang, Xing
AU - Cheng, Xiaodong
N1 - Funding Information:
We thank Yu Cao of MDACC for technical assistance. We thank Richard J. Roberts and Iain Murray of New England Biolabs for the gift of the anti-4mC antibody. We thank Xiangpeng Kong for assistance of access to 17-ID-1 beamtime. We thank the beamline scientists of Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source (APS), Argonne National Laboratory and 17-ID-1 of the National Synchrotron Light Source II, Brookhaven National Laboratory. The use of SER-CAT is supported by its member institutions and equipment grants ( S10_RR25528 , S10_RR028976 , and S10_OD027000 ) from the US National Institutes of Health . Use of the APS was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38. This research also used resources 17-ID-1 of the National Synchrotron Light Source II, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. The use of protein and monoclonal antibody production core at Baylor College of Medicine is supported by P30 Cancer Center Support Grant NCI-CA125123 .
Funding Information:
We thank Yu Cao of MDACC for technical assistance. We thank Richard J. Roberts and Iain Murray of New England Biolabs for the gift of the anti-4mC antibody. We thank Xiangpeng Kong for assistance of access to 17-ID-1 beamtime. We thank the beamline scientists of Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source (APS), Argonne National Laboratory and 17-ID-1 of the National Synchrotron Light Source II, Brookhaven National Laboratory. The use of SER-CAT is supported by its member institutions and equipment grants (S10_RR25528, S10_RR028976, and S10_OD027000) from the US National Institutes of Health. Use of the APS was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38. This research also used resources 17-ID-1 of the National Synchrotron Light Source II, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. The use of protein and monoclonal antibody production core at Baylor College of Medicine is supported by P30 Cancer Center Support Grant NCI-CA125123. J. Z. J. R. H. G. K. Q. C. X. L. F. M. T. W. and R. M. B. investigation; R. M. B. and X. C. writing–review and editing; X. Z. supervision; X. Z. and X. C. conceptualization; X. Z. project administration; X. C. funding acquisition; X. C. writing–original draft; X. C. methodology. The work was supported by US National Institutes of Health grant R35GM134744 (to X. C.), Cancer Prevention and Research Institute of Texas grant RR160029 (to X. C. who is a CPRIT Scholar in Cancer Research), and the Cockrell Foundation Fellowship (to J. Z.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
The work was supported by US National Institutes of Health grant R35GM134744 (to X. C.), Cancer Prevention and Research Institute of Texas grant RR160029 (to X. C., who is a CPRIT Scholar in Cancer Research), and the Cockrell Foundation Fellowship (to J. Z.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2023 The Authors
PY - 2023/8
Y1 - 2023/8
N2 - Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (A/C)CG(T/C/A), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle–regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle–regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis–dependent methylation following DNA replication.
AB - Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (A/C)CG(T/C/A), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle–regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle–regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis–dependent methylation following DNA replication.
KW - Adineta vaga
KW - DNA methylation
KW - N4-methylcytosine
KW - bdelloid rotifer
KW - metazoan cytosine-N4 DNA methyltransferase
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U2 - 10.1016/j.jbc.2023.105017
DO - 10.1016/j.jbc.2023.105017
M3 - Article
C2 - 37414145
AN - SCOPUS:85166634087
SN - 0021-9258
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 8
M1 - 105017
ER -