Promene strukture DNK izazvane proteinom HsOrc4
Structural changes of DNA mediated by HsOrc4 protein
Апстракт
U ćelijama svih živih organizama replikacija DNK se odvija u nekoliko složenih i međusobno veoma sličnih faza, a započinje tek kada inicijatorni proteinski kompleks prepozna specifične sekvence u genomu. I pored velikog biološkog značaja procesa replikacije malo se zna o mehanizmima koji regulišu inicicijaciju sinteze DNK višećelijskih eukariota. Ori sekvence ovih organizama ne sadrže konsenzus sekvence, ali su bogate AT baznim parovima grupisanim u kratke, naizmenične ili homogene nizove. Ovakvi elementi mogu da formiraju alternativne strukture, a one mogu biti značajne za interakciju ori sekvenci i inicijatornog kompleksa, ili mogu nastati tokom interakcije inicijacionog kompleksa i DNK i biti značajne za naredne faze inicijacije. U ovom radu su ispitivane interakcije rekombinantnog proteina HsOrc4 sa fragmentom izolovanim iz humanog replikatora LMNB2 i različitim sintetskim DNK koje sadžre A i T elemente. Protein je izabran za analizu jer čini deo kompleksa ORC (eng. Origin Recognit...ion Complex), pokazuje samostalnu vezivnu aktivnost i pripada familiji AAA+ superfamilije makromolekulskih šaperona. U radu je potvrđeno da se HsOrc4 preferencijalno vezuje za DNK bogatu AT baznim parovima i TAT elementima, i pokazano da stimuliše nekaknonsku renaturaciju komplementarnih fragmenata u specifičnu trolančanu strukturu. Takođe je pokazano da ovaj protein stimuliše i formiranje TAT tripleksa od odgovarajućih oligonukleotida. U radu je opisana i sasvim neočekivana sposobnost proteina da katalizuje asocijaciju homoadeninskih oligonukleotida u ranije nepoznate dvolančane i, verovatno, četvorolančane strukture. Homoadeninske strukture bile su zavisne od magnezijumovih jona, termostabilne i povezane Hugstinovim vezama. Sudeći prema delovanju mutiranih formi proteina HsOrc4 neaktivnih u vezivanju ili hidrolizi ATR-a, za katalitičku aktivnost HsOrc4 neophodno je vezivanje, ali ne i hidroliza ATR-a. Budući da je osnovna uloga inicijacionog kompleksa da remodeluje ori sekvencu i pripremi je za naredne faze inicijacije, opisano delovanje proteina HsOrc4 moglo bi da doprinese formiranju kompleksne arhitekture ori replikacije i da ima značajnu ulogu u inicijaciji replikacije.
In multicellular eukaryotes DNA replication initiates at fixed chromosomal sites called origins of replication (ori). They host initiation start sites and binding sites for universal eukaryotic initiator called origin recognition complex (ORC), but do not share conserved consensus sequences. All origins are AT rich and contain homopurine/homopyrimidine stretches that can form alternative DNA structures, potentially important for origin function. In order to understand alternative structures of origins and the manner in which specific proteins influence DNA structure, we have investigated interactions of recombinant protein HsOrc4 with various origin or synthetic DNAs. HsOrc4 was chosen as DNA-binding component of ORC and a member of the AAA+ superfamily of macromolecular chaperones. HsOrc4 preferentially recognized double-stranded and triple-stranded AT rich DNA and stimulated renaturation of the origin fragment lboI into specific triple stranded structure, or renaturation of compleme...ntary oligonucleotides into intermolecular TAT triplex. An unexpected feature of HsOrc4 shown in our study was its ability to catalyse self-association of adenines creating homoadenine duplexes and possibly quadruplexes. The action of HsOrc4 was dependant on the presence of magnesium ions and ATP. ATP had a role as a cofactor, because HsOrc4 did not require its hydrolysis for activity. The mutated form HsOrc4 WA, which could not bind ATP, was inactive in restructuring reactions, whereas mutated form HsOrc4 WB, unable to hydrolyse ATP, showed similar activity to the wild type HsOrc4. Ability to form Hoogsteen bonds was essential for substrates in restructuring reactions, as these bonds held newly formed structures together. Substrates containing 7-Deaza-ATP, dATP analogue unable to form Hoogsteen bonds, were inactive in these assays. The features of HsOrc4 shown in this study are potentially biologically important, because its action could expand structural repertoire of genomes by producing energetically unfavourable DNA structures. These structures may be involved in determination of origin specificity.
Кључне речи:
Struktura DNK / protein HsOrc4 / ORC (origin recognition) kompleks / ishodište (ori) replikacije / DNK tripleks / origin of replication / ORC complex / HsOrc4 protein / DNA triplex / DNA structureИзвор:
2008Издавач:
- Univerzitet u Beogradu, Biološki fakultet
URI
https://nardus.mpn.gov.rs/handle/123456789/10261http://eteze.bg.ac.rs/application/showtheses?thesesId=6314
https://fedorabg.bg.ac.rs/fedora/get/o:18975/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=33875983
https://imagine.imgge.bg.ac.rs/handle/123456789/12
Колекције
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - THES AU - Divac, Aleksandra PY - 2008 UR - https://nardus.mpn.gov.rs/handle/123456789/10261 UR - http://eteze.bg.ac.rs/application/showtheses?thesesId=6314 UR - https://fedorabg.bg.ac.rs/fedora/get/o:18975/bdef:Content/download UR - http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=33875983 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/12 AB - U ćelijama svih živih organizama replikacija DNK se odvija u nekoliko složenih i međusobno veoma sličnih faza, a započinje tek kada inicijatorni proteinski kompleks prepozna specifične sekvence u genomu. I pored velikog biološkog značaja procesa replikacije malo se zna o mehanizmima koji regulišu inicicijaciju sinteze DNK višećelijskih eukariota. Ori sekvence ovih organizama ne sadrže konsenzus sekvence, ali su bogate AT baznim parovima grupisanim u kratke, naizmenične ili homogene nizove. Ovakvi elementi mogu da formiraju alternativne strukture, a one mogu biti značajne za interakciju ori sekvenci i inicijatornog kompleksa, ili mogu nastati tokom interakcije inicijacionog kompleksa i DNK i biti značajne za naredne faze inicijacije. U ovom radu su ispitivane interakcije rekombinantnog proteina HsOrc4 sa fragmentom izolovanim iz humanog replikatora LMNB2 i različitim sintetskim DNK koje sadžre A i T elemente. Protein je izabran za analizu jer čini deo kompleksa ORC (eng. Origin Recognition Complex), pokazuje samostalnu vezivnu aktivnost i pripada familiji AAA+ superfamilije makromolekulskih šaperona. U radu je potvrđeno da se HsOrc4 preferencijalno vezuje za DNK bogatu AT baznim parovima i TAT elementima, i pokazano da stimuliše nekaknonsku renaturaciju komplementarnih fragmenata u specifičnu trolančanu strukturu. Takođe je pokazano da ovaj protein stimuliše i formiranje TAT tripleksa od odgovarajućih oligonukleotida. U radu je opisana i sasvim neočekivana sposobnost proteina da katalizuje asocijaciju homoadeninskih oligonukleotida u ranije nepoznate dvolančane i, verovatno, četvorolančane strukture. Homoadeninske strukture bile su zavisne od magnezijumovih jona, termostabilne i povezane Hugstinovim vezama. Sudeći prema delovanju mutiranih formi proteina HsOrc4 neaktivnih u vezivanju ili hidrolizi ATR-a, za katalitičku aktivnost HsOrc4 neophodno je vezivanje, ali ne i hidroliza ATR-a. Budući da je osnovna uloga inicijacionog kompleksa da remodeluje ori sekvencu i pripremi je za naredne faze inicijacije, opisano delovanje proteina HsOrc4 moglo bi da doprinese formiranju kompleksne arhitekture ori replikacije i da ima značajnu ulogu u inicijaciji replikacije. AB - In multicellular eukaryotes DNA replication initiates at fixed chromosomal sites called origins of replication (ori). They host initiation start sites and binding sites for universal eukaryotic initiator called origin recognition complex (ORC), but do not share conserved consensus sequences. All origins are AT rich and contain homopurine/homopyrimidine stretches that can form alternative DNA structures, potentially important for origin function. In order to understand alternative structures of origins and the manner in which specific proteins influence DNA structure, we have investigated interactions of recombinant protein HsOrc4 with various origin or synthetic DNAs. HsOrc4 was chosen as DNA-binding component of ORC and a member of the AAA+ superfamily of macromolecular chaperones. HsOrc4 preferentially recognized double-stranded and triple-stranded AT rich DNA and stimulated renaturation of the origin fragment lboI into specific triple stranded structure, or renaturation of complementary oligonucleotides into intermolecular TAT triplex. An unexpected feature of HsOrc4 shown in our study was its ability to catalyse self-association of adenines creating homoadenine duplexes and possibly quadruplexes. The action of HsOrc4 was dependant on the presence of magnesium ions and ATP. ATP had a role as a cofactor, because HsOrc4 did not require its hydrolysis for activity. The mutated form HsOrc4 WA, which could not bind ATP, was inactive in restructuring reactions, whereas mutated form HsOrc4 WB, unable to hydrolyse ATP, showed similar activity to the wild type HsOrc4. Ability to form Hoogsteen bonds was essential for substrates in restructuring reactions, as these bonds held newly formed structures together. Substrates containing 7-Deaza-ATP, dATP analogue unable to form Hoogsteen bonds, were inactive in these assays. The features of HsOrc4 shown in this study are potentially biologically important, because its action could expand structural repertoire of genomes by producing energetically unfavourable DNA structures. These structures may be involved in determination of origin specificity. PB - Univerzitet u Beogradu, Biološki fakultet T1 - Promene strukture DNK izazvane proteinom HsOrc4 T1 - Structural changes of DNA mediated by HsOrc4 protein UR - https://hdl.handle.net/21.15107/rcub_nardus_10261 ER -
@phdthesis{ author = "Divac, Aleksandra", year = "2008", abstract = "U ćelijama svih živih organizama replikacija DNK se odvija u nekoliko složenih i međusobno veoma sličnih faza, a započinje tek kada inicijatorni proteinski kompleks prepozna specifične sekvence u genomu. I pored velikog biološkog značaja procesa replikacije malo se zna o mehanizmima koji regulišu inicicijaciju sinteze DNK višećelijskih eukariota. Ori sekvence ovih organizama ne sadrže konsenzus sekvence, ali su bogate AT baznim parovima grupisanim u kratke, naizmenične ili homogene nizove. Ovakvi elementi mogu da formiraju alternativne strukture, a one mogu biti značajne za interakciju ori sekvenci i inicijatornog kompleksa, ili mogu nastati tokom interakcije inicijacionog kompleksa i DNK i biti značajne za naredne faze inicijacije. U ovom radu su ispitivane interakcije rekombinantnog proteina HsOrc4 sa fragmentom izolovanim iz humanog replikatora LMNB2 i različitim sintetskim DNK koje sadžre A i T elemente. Protein je izabran za analizu jer čini deo kompleksa ORC (eng. Origin Recognition Complex), pokazuje samostalnu vezivnu aktivnost i pripada familiji AAA+ superfamilije makromolekulskih šaperona. U radu je potvrđeno da se HsOrc4 preferencijalno vezuje za DNK bogatu AT baznim parovima i TAT elementima, i pokazano da stimuliše nekaknonsku renaturaciju komplementarnih fragmenata u specifičnu trolančanu strukturu. Takođe je pokazano da ovaj protein stimuliše i formiranje TAT tripleksa od odgovarajućih oligonukleotida. U radu je opisana i sasvim neočekivana sposobnost proteina da katalizuje asocijaciju homoadeninskih oligonukleotida u ranije nepoznate dvolančane i, verovatno, četvorolančane strukture. Homoadeninske strukture bile su zavisne od magnezijumovih jona, termostabilne i povezane Hugstinovim vezama. Sudeći prema delovanju mutiranih formi proteina HsOrc4 neaktivnih u vezivanju ili hidrolizi ATR-a, za katalitičku aktivnost HsOrc4 neophodno je vezivanje, ali ne i hidroliza ATR-a. Budući da je osnovna uloga inicijacionog kompleksa da remodeluje ori sekvencu i pripremi je za naredne faze inicijacije, opisano delovanje proteina HsOrc4 moglo bi da doprinese formiranju kompleksne arhitekture ori replikacije i da ima značajnu ulogu u inicijaciji replikacije., In multicellular eukaryotes DNA replication initiates at fixed chromosomal sites called origins of replication (ori). They host initiation start sites and binding sites for universal eukaryotic initiator called origin recognition complex (ORC), but do not share conserved consensus sequences. All origins are AT rich and contain homopurine/homopyrimidine stretches that can form alternative DNA structures, potentially important for origin function. In order to understand alternative structures of origins and the manner in which specific proteins influence DNA structure, we have investigated interactions of recombinant protein HsOrc4 with various origin or synthetic DNAs. HsOrc4 was chosen as DNA-binding component of ORC and a member of the AAA+ superfamily of macromolecular chaperones. HsOrc4 preferentially recognized double-stranded and triple-stranded AT rich DNA and stimulated renaturation of the origin fragment lboI into specific triple stranded structure, or renaturation of complementary oligonucleotides into intermolecular TAT triplex. An unexpected feature of HsOrc4 shown in our study was its ability to catalyse self-association of adenines creating homoadenine duplexes and possibly quadruplexes. The action of HsOrc4 was dependant on the presence of magnesium ions and ATP. ATP had a role as a cofactor, because HsOrc4 did not require its hydrolysis for activity. The mutated form HsOrc4 WA, which could not bind ATP, was inactive in restructuring reactions, whereas mutated form HsOrc4 WB, unable to hydrolyse ATP, showed similar activity to the wild type HsOrc4. Ability to form Hoogsteen bonds was essential for substrates in restructuring reactions, as these bonds held newly formed structures together. Substrates containing 7-Deaza-ATP, dATP analogue unable to form Hoogsteen bonds, were inactive in these assays. The features of HsOrc4 shown in this study are potentially biologically important, because its action could expand structural repertoire of genomes by producing energetically unfavourable DNA structures. These structures may be involved in determination of origin specificity.", publisher = "Univerzitet u Beogradu, Biološki fakultet", title = "Promene strukture DNK izazvane proteinom HsOrc4, Structural changes of DNA mediated by HsOrc4 protein", url = "https://hdl.handle.net/21.15107/rcub_nardus_10261" }
Divac, A.. (2008). Promene strukture DNK izazvane proteinom HsOrc4. Univerzitet u Beogradu, Biološki fakultet.. https://hdl.handle.net/21.15107/rcub_nardus_10261
Divac A. Promene strukture DNK izazvane proteinom HsOrc4. 2008;. https://hdl.handle.net/21.15107/rcub_nardus_10261 .
Divac, Aleksandra, "Promene strukture DNK izazvane proteinom HsOrc4" (2008), https://hdl.handle.net/21.15107/rcub_nardus_10261 .