Interakcija inicijacionog proteina ORC sa replikatorom regiona lamin B2

Abstract

Inicijacija replikacije DNK kod eukariota započinje vezivanjem proteinskog kompleksa ORC za diskretna mesta u genomu nazvana ori-sekvence ili ori. Specifične sekvence odgovome za prepoznavanje i vezivanje kompleksa ORC, kao i mehanizam kojim ORC selektuje orisekvence još uvek nisu poznati. Jedna od malobrojnih zajedničkih karakteristika mesta uključenih u vezivanje inicijacionog proteina jeste visok sadržaj AT bp i kratkih nizova (dA)-(dT) što ukazuje na mogućnost da ori-sekvence obrazuju sinonimne stmkture koje odreduju specifičnost vezivanja inicijacionog proteina. Radi testiranja ove pretpostavke, analizirana je struktura i oblik humane ori-sekvence lamin B2 i detaljno ispitana DNK vezivna specifičnost proteina HsOrc4. Ovaj protein predstavlja jednu od subjedinica kompleksa ORC i pokazuje istu DNK-vezivnu aktivnost kao i kompleks ORC. Utvrđeno je da ori-sekvenca, pod uslovima neutralnog pH, niske ili umerene jonske jačine i u prisustvu jona Mg2+ zauzima altemativnu formu koju karakteriše prisustvo denaturisanog regiona, okceta, i povećana elektroforetska pokretljivost u nativnoj PAGE. Stabilno prisustvo denaturisanog regiona u okviru fragmenta DNK omogućeno je formiranjem Hoogsteen-ovih vodoničnih veza izmedu centralnih pirimidina okceta i adenina jednog od kratkih nizova (dA)-(dT). Kao rezultat ove interakcije nastaje dvolančana petlja koja u osnovi sadrži intramolekulski tripleks. Struktura intramolekulskog tripleksa formira se u regionu koji interaguje sa inicijacionim proteinom i može predstavljati jedan od elemenata odgovornih za prepoznavanje i vezivanje ovog proteina. U eksperimentima direktnog vezivanja proteina HsOrc4 utvrdeno je da on ne prepoznaje kratke nizove (dA)-(dT) i (dA), dok se za (dT) slabo vezuje. Najveći afmitet protein je pokazivao za trolančane strukture tipa TAT. Kompeticija jedno-, dvo- i trolančanih molekula DNK vezivanju HsOrc4 za ori-sekvencu pokazala je da protein razlikuje trolančanu strukturu od jedno- i dvolančane i specifično se vezuje za nju. Sudeći po efikasnosti kompeticije, trolančana DNK bila je veoma slična prirodnim vezivnim mestima proteina HsOrc4. Svojstvo ori-sekvence da formira intramolekulski tripleks i specifično vezivanje proteina HsOrc4 za tripleks sugeriše da trolančana struktura može predstavljati deo mehanizma kojim inicijacioni protein prepoznaje i selektuje mesta inicijacije replikacije.

In complex eukaryotes DNA replication is initiated by binding of origin recognition complexes (ORCs) to specific genomic sites called origins of replication. Consensus sequence required for this event and the mechanism by which ORC is localized to origins of replication remain poorly understood. General features of genomic regions involved in initiator protein binding are AT-richness and frequent occurrence of short (dA)-(dT) runs. Such distribution of A and T residues opens a possibility that origins of replication build mutually equivalent unorthodox structures which are recognised by initiation protein. In order to test this hypothesis, a study of structure and shape of the human lamin B2 origin was performed. DNA binding activity of protein HsOrc4, one of ORCs subunits that exhibited similar DNA binding properties as the whole complex, was also tested. It was shown that, at neutral pH, low or moderate ionic strength and in presence of Mg" ions, lamin B2 ongm adopted alternative helical form, characterized by a single unpaired region and faster migration in native polyacrylamide geis. It was proposed that these properties reflected the ability of origin DNA to form double stranded loop with intramolecular triplex in its base. Triplex was kept together by Hoogsteen hydrogen bonding between Central pyrimidines of unpaired region and complementary double stranded sequence. Since intramolecular noncanonical structure formed in origin sequences protected by ORC in vivo and in vitro, it could represent the element responsible for site-specific ORC binding. In order to test this notion, binding specificity of HsOrc4 was tested in direct and competition DNA binding experiments. The protein did not recognize (dA) or (dA)-(dT), but it exhibited very low affmity for (dT) and a very high affmity for TAT triplex. Consistent with that, triple stranded DNA competed very well with origin DNA for HsOrc4 binding, whereas single or double stranded DNA exhibited much less significant competitive effect. As judged by its competitive efficiency, triple stranded DNA was very similar to naturally ocuring DNA binding sites of HsOrc4. In conclusion, formation o f intramolecular triplex within origin DNA and its specific recognition by HsOrc4 suggest that triple stranded structure might play a role in selection of eukaryotic origins of replication.