C ch phn t ca qu trnh ti bn DNANhng nghin cu tip theo tm ra c ch
phn t ca qu trnh ti bn. l mt qutrnh phc tap c su khc bit prokaryote
v eukaryote nhung phi tri qua c ch chungsau: Cc lin kt hydrogen n
jnh cu trc xon v lin kt hai mach n vi nhau phi uoc ph v tch ri hai
mach. Phi c oan mi (primer), tc l oan DNA/RNA ngn, bt cp b sung vi
mach khun tao u 3'OH tu do. Nguyn liu tng hop DNA l cc
desoxynucleosid 5'-triphosphate (dNTPs): dATP, dGTP,dCTP v TTP.
Mach khun lun uoc oc theo chiu 3'-5' trong khi mach mi uoc tng hop
theo chiu 5'-3'. Mi nucleotide mi uoc gn vo u 3'OH ca mach ang ko
di bng lin kt cng ho trj. Nng luong cho su polymer ho n t vic thy
phn cc dNTPs, loai ra cc pyrophosphate. Mi buc uoc thuc hin mt cch
nhanh chng, chnh xc dui su iu khin ca enzyme c hiu.n v ti bn, im
khi u v im kt thc ti bnMi oan DNA uoctibn nhu l mt n vj ring l uoc
goilmtn vjtibn(replicon). Cc DNA dang vng, kch thuc nh (chng han
nhu nhim sc th ca vi khunv DNA virus) chi gm mt n vj ti bn duy nht.
Su ti bn khi u t mt im goi lim khi u sao chp (origin) v lan ra theo
hai hung, hnh thnh hai chac ba ti bn(replication fork) cho n khi gp
nhau tai im kt thc (terminus). im khi u ti bn ckhuynh hung giu A-T
d dng khi u tch ri hai mach n. eukaryote, mi phn t DNA bao gm nhiu
n vj ti bn. Mi n vj ti bn c imkhi u ring v su ti bn cng lan ra theo
hai hung. Khi cc chac ba ti bn gp nhauth su ti bn DNA uoc hon thnh.
V du nhu t bo hu nh in hnh c t 50.000 100.000 n vj ti bn, mi n vj
ti bn c kch thuc khong 40 200 kb.1.4 Su tng hop mach mi ca DNA xy
ra lin tuc trn mt mach v gin oantrn machkia(Semi-discontinuous
replication - Ti bn bn gin oan)Trong ti bn bn bo tn, c hai mach mi
ca DNA uoc tng hop ng thi mi chacba ti bn. Tuy nhin, c ch ti bn DNA
chi cho php su tng hop theo hung 5' 3', vth trn haimach khun c hung
nguoc nhau, su tng hop mach mikhng din tinging nhau. T im khi u ti
bn, trn mach khun 3'- 5' su tng hop mach mi din ra mt cch lin tuc
theo chiu 5'- 3' cng chiu vi hung tho xon. Mach ny uoc goi l mach
ti (leading strand). Trong khi trn mach khun 5' 3', su tng hop mach
mi cng din ra theo hung 5' 3' nhung nguoc vi hung tho xon. Chnh v
th, su tng hop mach mi khng xy ra lin tuc m dui dang nhng oan ngn
uoc goi l oan Okazaki (kch thuc 1000 2000 bp prokaryote v 100 200
bp eukaryote). Mach mi uoc tng hop theo kiu gin oan ny uoc goi l
mach chm (lagging strand).Trn thuc t, su tng hop c hai mach theo
cng hung v mach khun chm uoc unvng quay 180o tai chac ba ti bn, tr
nn cng hung vi mach khun ti.Mi RNAMach tiv tt c nhng oan Okazaki ca
mach chm chi c th uoc tng hop bngcch ko di mt mi bt cp sn trn mach
khun. Mi l mt oan RNA ngn, uoctng hop bi mt phc hop protein goi l
primosome bao gm nhiu protein v mt enzymec tn l primase. V du, E.
coli, phc hop primosome bao gm cc Dna B helicase (tchri hai mach n)
v DNA primase (tng hop mi RNA t khun DNA).Cc mi RNA ny sau s bj phn
hy bi RNase v uoc thay bng trnh tu DNA nhvo hoat ng ca DNA
polymerase. Enzyme ligase s ni lin cc oan DNA lai vi nhau.C ch dng
mi l RNA thay v DNA dung nhu lin quan n tnh chnh xc cao trong tibn
DNA v hoat tnh sa sai ca cc DNA polymerase.Ti bn DNA prokaryoteCc
nghin cu v ti bn DNA uoc tin hnh dua trn cc h thng th nghim in
vitro. theo di ti bn DNA, ngui ta dng thymidine H3phng xa. Qu trnh
ti bn xut pht tim khi u v lan ra hai pha. Khi quan st DNA vng trn
ang ti bn, ta thy dangDNA "hnh con mt (eye replication). Hai chac
ba ti bn lan dn, cui cng tao thnh haiphn t DNA lai, trong c mt mach
mang du phng xa T-H3. C trung hop, su ti bnchi xy ra v mt
pha.Dinbinti bnnhimscthvngtrncthchialmbagiai oan: khi
u(initiation), ko di (elongation), kt thc v phn chia (termination
& segregation).Giai on khi uTho xon phn t DNAM hnh phn t ca DNA
vng vi khun hay virus cho ta mt khi nim chung vcc cu hnh c th c ca
mt phn t DNA. Dng th nht l siu xon. Dng thhai c cu trc li hn, thng
l do b t mt ch trn mt trong hai mch ca phnt DNA. Dng th ba tng ng
vi cu trc thng do s ct t trn c hai mchtrong phn t DNA. Trong , dng
siu xon l dng c bn v cu trc v chcnng.Nh vy, bt u ti bn, phn t DNA
phi c tho xon nh vo hot ng cacc enzyme c tn l topoisomerase. C hai
loi topoisomerase. Loi I tho dng siuxon, chng gn vo phn t DNA v ct
mt trong hai mch. Sau khi gii phng mtphn t DNA c tho xon, cc enzyme
ny s ni li ch t. Enzyme loi Ic bit r nht l protein ca E. coli. Cc
topoisomerase loi II c kh nng thocc nt ny sinh do cc bin i cu trc
ca chui xon kp bng cch ct t c haimch DNA. Enzyme c bit r nht l
gyrase ca E. coli. Gyrase s dng nnglng t s thy phn ATP tho xon
DNA.Tch ri hai mch n ti im khi u ti bn E.coli, OriC cha bn v tr gn
vi protein khi u c tn l Dna A. Mi v tr ckch thc 9bp. S tng hp cc
protein ny gn lin vi tc tng trng t bo vth vic khi u ti bn DNA cng
gn lin vi tc tng trng. tc tngtrng cao, cc nhim sc th ca vi khun c
th bt u ln ti bn th hai trckhi ln ti bn th nht kt thc ti hai im khi
u mi. V vy, mi t bo con snhn c mt nhim sc th ang c ti bn mt phn.Qu
trnh ti bn bt u khi DNA OriC qun quanh mt phc hp protein Dna A
gm30-40 phn t, mi phn t gn vi mt ATP. iu ny thc y s tch ri haimch
ti ba trnh t lp 13bp giu A T, cho php protein Dna B gn vo. Dna B
lmt DNA hehicase, nm trong phc hp primosome. Cc helicase ph v lin
kthydro gia cc base nh nng lng thy phn ATP. C nhiu loi helicase cng
hotng ng thi: Mt s gn trn mch 3 5 nh cc Rep, s khc gn trn mch 5 3
nh helicase II v III.Cc mch tch ri s c n nh di dng mch n nh cc
protein SSB(Single Strand Binding lin kt vi mch n). Cc protein ny
gn ln khp phnmch n lm cho hai mch khng kt hp tr li c. Mch khun c s
dngn u th cc protein SSB c gii phng khi khun n .Giai on ko diTng hp
mi RNACc DNA polymerase ch c th tng hp DNA bng cch ko di mt mi RNA
btcp sn trn khun. Nh bit, mi ny c tng hp nh hot tnh ca
enzymeprimase c trong phc hp primosome.S tng hp mch mi din ra theo
kiu bn gin on, ko di mi RNADNA polymerase III l mt phc hp dimer. Mi
phn gm nhiu n v gn vi nhau,chu trch nhim tng hp mt mch n DNA nhm m
bo cho tc tng hpca c hai mch bng nhau. n v c hot tnh polymerase tht
s. n v cchc nng c sa nh hot tnh exonuclease 3- 5, t lm tng tnh chnh
xctrong ti bn. n v gip gn polymerase vo DNA. y l nhn t duy tr,
khcnhau hai mch khun, quy nh di ca on DNA c tng hp mch ti(di) khc
vi mch chm (ngn).DNA polymerase III bt u ti bn trn mi mch khun bng
cch gn vo mch(nh n v ) v lp cc nucleotide b sung vo v tr tng ng, ko
di on miRNA bt cp sn trn khun t u 3OH t do ca mi (nh n v ). Cc
DNApolymerase c tnh c hiu cao, ch thm nucleotide vo u 3OH ca mch
angtng hp.Ngoi chc nng polymer ha theo hng 5 - 3, DNA polymerase
III cn c kh nngsa sai nh hot tnh exonuclease theo hng 3- 5.
Exonuclease l hot tnh enzymect DNA t u mt mt mch. Trn ng di chuyn
tng hp mch mi, nu gpch nucleotide va lp sai v tr, DNA polymerase
III s dng hot tnh exonuclease 3-5 ct li li b nucleotide sai v lp ci
ng vo ri tip tc ti bn (n v ).Qu trnh ti bn DNA E.coli din ra vi tc
nhanh, c th n 50.000nucleotide/pht.Hon chnh si mi tng hpTrn mch
chm, sau khi mi on DNA c ko di, mi RNA b di i nh hottnh exonuclease
53 ca DNA polymerase I v s b enzyme RNase H phn hy. Ccch trng m mi
li s c thay bng trnh t DNA mt cch chnh xc nh kthp hot tnh
polymerase 5 3 (ko di u 3 ca on Okazaki trc) vi hot tnhexonuclease
3 5 (c v sa sai) ca DNA polymerase I. Cui cng, ligase s nitt c cc
on DNA trn mch mi tng hp li vi nhau.Giai on kt thc ti bn v phn chia
t boHai chc ba ti bn s gp nhau khong 180o i din vi OricC. Quanh vng
ktthc ny c vi im lm dng li s ti bn bng cch gn vi mt sn phm cagen
tus, l mt nhn t km hm hot ng helicase ca Dna B.Khi s ti bn hon tt,
hai phn t DNA vng vn cn dnh vi nhau. Mttopoisomerase loi II c tn l
topoisomerase IV s tch ri chng sau hainhim sc th con s c phn phi vo
hai t bo con.Ti bn DNA eukaryoteSu ti bn eukaryote vn cn nhng iu
chua tung tn nhung cc d liu thu uoc chothy h thng ny kh gn vi h
thng ti bn prokaryote. Khc bit ch yu l cc loaiDNA polymerase tham
gia vo qu trnh.im khi u ti bn v giai on khi u:V su phc tap trong cu
trc ca cht nhim sc, tc di chuyn ca cc chac ba ti bn eukaryote chi
at khong 50 bp/giy. Vi tc ny, nu chi s dung hai chac ba ti bn,phi
mt 30 ngy mi ti bn xong mt phn t DNA nhim sc th in hnh ca ng vthu
nh vi kch thuc 105 kb. Do cn phi c nhiu n vj ti bn trong mt t bo,
inhnh nhu ng vt hu nh l 50.000 100.000 n vj ti bn. eukaryote, c
khong 20 50 n vj ti bn khi u cng lc tai mi thi im, xuynsut phase S.
Phn ti bn smnht ch yu bao gmnguyn nhimsc cht(euchromatin), tc l phn
DNA c hoat ng phin m. Trong khi , djnhim sc cht(heterochromatin)
uoc hoat ha mun hn v phn DNA ca tm ng (centromere) voan cui nhim sc
th (telomere) uoc ti bn sau cng. iu ny phn nh kh nngp ng khc nhau
ca nhng cu trc nhim sc cht vi nhn t khi u.Cc trnh tu khi u ng vt hu
nh chua uoc phn lp nhung ngui ta tin rng sukhi u ca mi n vj ti bn c
l din ra ty bn trong mt vng khi u c th din vi kb v l mt phn ca trnh
tu DNA lp lai trung bnh.Khc vi prokaryote, cc n vj ti bn ca
eukaryote chi khi u mt ln trong mi chutrnh t bo. Mt protein goi l
nhn t cho php (licensing factor) cn thit cho su khi uv ti hoat ha
chi c kh nng tip cn vo nhn khi mng nhn tan i, v th ngn chnuoc vic
ti khi u truc han jnh.Chc ba ti bn v h thng cc enzim ti bnTruc khi
ti bn, DNA phi uoc tho khi nucleosome tai mi chac ba ti bn. iu ny
lm chm su di chuyn ca chac ba, chi cn 50 bp/giy. Sau khi chac ba ti
bn i qua,cc nucleosome uoc tilp rp t cc histone c v histone miuoc
tng hop. Kchthuc nh ca cc oan Okazaki (v du 135 bp SV40) phn nh
luong DNA uoc thokhi mi nucleosome khi chac ba ti bn i qua. Ngoi su
nhn i DNA, cc histone cngphi uoc nhn ln gp i trong sut phase S.H
thng cc DNA polymerase eukaryote phc tap hn so vi prokaryote, bao
gm: Polymerase d/primase: C chc nng tng hop mi cho mach ti v cho c
nhng oan Okazaki ca mach chm. Polymerase ny tip tuc ko di DNA nhung
nhanh chng bj thay bi polymerase trn mach ti v polymerase s trn
mach chm. Polymerase d khng c hoat tnh exenuclease. Polymerase : C
chc nng ging DNA polymerase prokaryote, ngha l tng hop ikm vi sa
sai v hon chinh mach mi sau khi mi RNA uoc loai b. Polymerase v
polymerase s : C chc nng ko di DNA. Trong kh nng tng hop oan DNA di
nht thuc v polymerase vi su tro gip ca PCNA. C hai enzyme ny u c kh
nng oc v sa sai. : uoc tm thy trong ti th, chc nng chua
r.Polymerase Ngoi cc polymerase k trn, h thng ti bn eukaryote cn c
su tham gia ca nhiuprotein chuyn bit nhu PCNA (Proliferating Cell
Nuclear Antigen - khng nguyn trong tbo ang phn chia) c chc nng hoat
ha cc polymerase v s, cc nhn t ti bn A vC (Replication actor, R -A,
R - C) cn cho hoat ng ca cc polymerase d v ...Ti bn on cui nhim sc
th (telomere replication):Hai oan cui ca nhim sc th dang thng khng
th uoc ti bn y bi v khng thko di DNA t u 5' ca mach mi tng hop sau
khi loai b mi RNA. Do thng tindi truyn s mt dn khi DNA sau mi ot ti
bn. gii quyt vn ny, oan cui mi nhim sc th eukaryote bao gm hng trm
bnsao mt trnh tu n gin, khng m ha (v du nhu TTAGGG ngui) vi u 3' nh
ra sovi u 5'.Telomerase l mt enzyme duy tr di ca telomere. Enzyme
ny c cha mt phn tRNA ngn. Mt phn trnh tu ca RNA ny b sung vi trnh
tu lp ca telomere, do nc thhoatng nhu mtmach khun ko dicc trnh tu
lp t u 3' tudo catelomere bng cch tng hop ri djch chuyn lin tip
nhiu ln.Cc eukaryote n bo, chng han nhu nm men, nh vo hoat ng ca
telomerase mduy tr telomere, t bo v t bo khi su mt thng tin di
truyn. Th nhung cc sinhvt eukaryote a bo, cc t bo sinh dung, gen m
ha telomerase bj km hm. V th,qua mi ot phn bo, cc nhim sc th ngn dn
i cho n khi cham ti cc trnh tu mha ca DNA, t bo s gi i v cht. Trong
nui cy t bo sinh dung, hu ht cc t bochi phn chia 30-40 hoc 50 ln ri
cht. cc t bo ung thu, su ti hoat ha hoat ng ca telomerase khin cho
cc t bo nyc kh nng bt t.Mt trong nhng tnh cht cn bn ca DNA l kh nng
tu sao chp (replication) hay l tunhn i (self duplication). Ngha l t
mt phn t DNA m sao chp tao ra hai phn tDNA con ging y nhu phn t DNA
m ban u.Su ti bn DNA l mt trong nhng tnh cht quan trong, nh m thng
tin di truyn uoctruyn t th h ny sang th h khc.L thuyt v sao m DNA
theo khunGi thuyt ca Watson - CrickSau khi xy dung m hnh cu trc ca
phn t DNA, atson v Crick thy rng, nu haisoi DNA tch ri ra th mt soi
s lm khun tng hop mt soi mi v do cc baz nitlun c xu hung bt cp vi
nhau (A viT v G vi C). Theo du on ca atson vCrick, qu trnh tng hop
DNA c th tm tt nhu sau- u tin lin kt hydr s bj t ra tao hai mach n
lm khun.- Cc baz nit mi s bt cp b sung vi cc baz nit trn soi lm
khun theo qui tc Ai din vi T v G i din vi C.- Kt thc qu trnh sao m,
t mt soi DNA xon kp m s tao hai soi xon kp con. Misoi xon kp con c
mt soi mi tng hop v mt soi s lm khun.Ngay sau khi m hnh uoc nu ra,
nhiu th nghim chng minh uoc tin hnh xcnhn du on.Th nghim chng minh
ca Meselson - Stahl (1958)Th nghim ny nhm muc ch chng minh kiu tng
hop theo khun ca DNA nhu duon ca atson v Crick. Th nghim uoc thuc
hin trnE. Coli. Vi khun uoc nui cyqua nhiu th h trn mi trung c cha
nit ng vj nng N15 v nit thung N14 .Nguyn tc ca th nghim l dua trn
kh nng phn bit ti trong ca hai ng vj N15 vN14 c mt trong cc loai
DNA khi li tm trn thang nng CsCl2 . Qu trnh th nghimuoc tin hnh nhu
sau- Th h u (goi l th h 0): vi khun uoc nui trn mi trung cha N15
nhu vy thh ny, cc nguyn t nit trong DNA ca vi khun s l N15 . Sau
khi nui tin hnh tchDNA v em li tm trn thang nng CsCl2, nhn thy th h
0: chi cha 1 gii n c ti trong cao. Nhu vy tt c DNA ca th h cha m
ucha 2 soi vi nit nng.- Th h 1: Chuyn vi khun th h 0 sang nui trn
mi trung cha N14. Thi gian viu kin nui tung tu nhu th h 0. Sau khi
tch DNA v em li tm nhu th h 0, ththy th h 1 chi cha 1 gii n c ti
trong trung bnh. Nhu vy mi phn t con th h 1c 1 soi cha nit nng ca b
m v 1 soi b sung mi tng hop c cu tao t nit nhN14, goi l soi DNA
lai.- Th h 2: Tip tuc chuyn vikhun ca th h 1 sang th h 2 v cng
nuitrn mitrung cha N14. Qu trnh th nghim tin hnh tung tu nhu cc th
h trn. Sau khi litm, quan st thy c mt na s luong phn t DNA l phn t
lai ging th h 1 v mtna s luong cn lai l phn t DNA vi c hai soi u
mang N14M hnh th nghim ca Meselson-StahTh nghim trn l mt bng chng
chng minh gi thuyt ca atson v Crick l ng.Qu trnh tng hop DNA phi
thuc hin theo khun, hai mach ban u uoc tch ra, mi cilm khun tng hop
mach mi. Kt qu l, mi phn t con u mang mt mach c vmt mach mi. Kiu
sao m nhu vy goi l sao m bn bo tn (semi-conservative).Th nghim chng
minh ca Arthur v KornbergCng trong nm ny (158), Arthur v Kornberg
tai trung ai hoc ashington tinh sachmt loai enzyme t E. Coli, goi l
DNA-polymerase. Enzyme ny xc tc tng hop mt soipolynucleotide t cc
nucleotide-3- phosphat.Nguyn liu dng cho th nghim gm- Bn loai
nucleotide dang triphosphat: dATP, dTTP, dGTP, dCTP.- Enzyme
DNA-polymerase chit t vi khun E.Coli.- on Mg2 l cofactor ca enzyme,
tao iu kin thun loi cho hoat ng ca enzyme.Tin trnh th nghim c th m
t nhu sauTh nghim th nht uoc tin hnh vi y cc nguyn liu nhu gii thiu
trn,trong cc iu kin thun loi cho su tng hop soi DNA mi, nhung khng
c soi lm khun.Th nghim th hai cng uoc tin hnh tung tu nhu th nghim
1 nhung c mt soi DNAlm khun. Soi lm khun uoc thu nhn bng cch lm bin
tnh v tch mach, sau ua vo th nghim.Ho thy rng, th nghim th nht,
enzyme DNA-polymerase khng th tng hop soi DNAt cc nguyn liu khi
khng c mt ca soi khun, vic tng hop soi DNA mi chi xy ra th nghim th
hai, khi c mt DNA khun. iu khng jnh rng gi thuyt ca atsonv Crick l
ng. DNA uoc tng hop chi khi c mt soi lm khun.Qu trnh sao mCc enzyme
v protein c hiu tham gia sao my l mt qu trnh phc tap, thuc hin sao
m cn thit phi c mt ca cc enzyme,cc protein c hiu v cc iu kin cn
thit sau y:- in kt hydro gia 2 mach b sung ca soi DNA m phi bj ph v
v tch ri tng buclm 2 mach lm khun.- Phic oan mi(primer), tc oan RNA
mach n ngn bt cp vimach n DNAkhun t vj tr bt u ca mi oan sao chp.-
C 4 loai nucleotide dang triphosphat (dATP, dTTP, dGTP, dCTP) bt cp
b sungvi cc nucleotide mach khun.- Mach mi uoc tng hop theo hung
5'P 3'OH.- Mi buc uoc iu khin bi enzyme c hiu v uoc thuc hin mt cch
nhanh chng,chnh xc.- C mt ca ion Mg2 lm cofactor ca enzyme.- Trong
qu trnh sao chp, ngoi cc enzyme c hiu cn c cc protein c hiu.Cc
enzyme v protein c hiu tham gia sao chp gm c- Enzyme topoisomerase,
lm nhim vu tho xon v hai pha trn soi DNA kp- Enzyme helicase, s
dung nng luong ATP lm t lin kt hydr gia cc baz nitca hai mach
khun.- DNA-polymerase , -, - lm nhim vu tng hop soi mi v sa sai.-
RNA-polymerase, lm nhim vu tng hop oan mi.- Enzyme primase, lm nhim
vu gn mi.- Enzyme ligase, lmnhimvu ni hai nucleotide ng canh nhau
bng lin ktphosphodiester tao mach polynucleotide.- Enzyme
ribonuclease (RNase) lm nhim vu ct b mi sau khi tng hop xong mach
mi.Sao chp theo quy lut b sungCc protein tham gia sao m gm c-
Protein B, nhn bit im khi u (origin) sao chp trn soi DNA kp.-
Protein SSB (Single Strand Buiding), lm nhim vu gi hai mach ca soi
DNA khng chochp vo nhau, tao iu kin thun loi cho qu trnh sao chp.Qu
trnh sao mNhn bit im khi s v tho xon DNAQu trnh sao m uoc nghin cu
kh k t bo vi khun E.Coli. Su sao m bt u khiprotein B c hiu nhn bit
im khi su sao m (replication origine) v gn vo trnh tuc hiu . Tip
theo, enzyme topoisomerase thuc hin tho xon phn t DNA t imkhi
su.Enzyme helicase s dung nng luong ATP ct t cc lin kt hydro gia cc
baz bt cpca hai mach phn t, tch hai mach tao thnh chac ba hnh ch ,
goi l chac ba saom (replication fork). C nhiu loai enzyme helicase:
c loai gn trn mach, di chuyn v ctlin kt hydro theo chiu t u 3 n u 5
c loai gn ln mach, di chuyn v ct lin kthydro theo chiu 5 3. Sau khi
tch ri, hai mach n s uoc protein lm cng machSSBgikhng cho chplai,
lmchotrangthi mxon uocbnvng.Mi phn tprotein SSB bm vo 8 nucleotide
trn mach n, mi chac ba sao m c khong 250 phnt protein SSB hoat ng.
Mach khun uoc s dung n u, cc phn t protein SSB suoc gii phng n .Tng
hp miEnzyme DNA-polymerase chi c kh nng tng hop soi DNA mibng cch
nidiu3OH tu do ca mt oan mi bt cp sn trn khun theo chiu 3OH n u
5P.Mi l mt oan RNA nh chng 10 nucleotide, uoc tng hop t khun ca soi
DNA. Phchop enzyme primase- RNA-polymerase bm vo mach n ca chac sao
m tng hop oanRNA mi tao u 3OH tu do ca oan mi. Enzyme
DNA-polymerase xc tc tng hopmach b sung t u 3OH tu do ca mi, ko di
mach. Su tng hop cc mach n DNAmi, v vy, chi i theo mt chiu xc jnh t
u 5 n u 3 (53).S tng hp mch DNA mi xy ra mt cch lin tc trn mch khun
c chiu 3' 5' v gin on trn mch khun c chiu 5' 3'Trn mach khun c chiu
35, mach mi uoc tng hop theo chiu 53 mt cch lintuc, cng hung tho
xon ca phn t DNA. oan mi s uoc tng hop t im khi susao m. Khi xut
hin u 3OH tu do ca oan mi th enzyme DNA-polymerase gnvo v tng hop
ngay mach b' sung theo chiu 53 mt cch lin tuc cho n im ktthc sao m.
Mach ny uoc tng hop nhanh hn nn ngui ta thung goi l mach
nhanh(leading strand). Nhu vy, tng hop uoc soi DNA mi b sung cho
soi lm khun ny,enzyme DNA-polymerase chi cn c mt oan miS biu din
cch sao chp gin oan v lin tuc trn 2 soi DNA khunTrn mach khun c
chiu t u 53, vic tng hop mach mi phc tap hn, do qui luttng hop soi
DNA mi b sung lun thuc hin theo chiu t u 53. Mach mi b sungcho mach
khun ny uoc tng hop duidang tng oan ngn goil okazaki. Mimtokazaki
cmt oanmi. vi khun, di mt okazaki khong 1.000n2.000nucleotide, cn t
bo eucaryote, s luong nucleotide trong mt okazaki ngn hn so vivi
khun. Hungdi chuyntnghopsoi mi caDNA-polymerase nguocvihung tho xon
ca phn t DNA m.Soi con th hai ny uoc tng hop mt cch chm hn nn thung
goi l mach chm haymach sau (lagging strand). Cc oan RNA mi sau s bj
enzyme ribonuclease phn hu.Cc l trng xut hin sau khicc oan mimt is
uoc lp y nh hoat ng caenzyme DNA-polymerase . Cui cng, enzyme
DNA-lygase ni cc lin kt phosphodiestergia cc oan, tao nn mach DNA
con hon chinh.Ngoi chc nng tng hop soi DNA mi theo chiu t u 53,
enzyme DNA-polymerase cn c kh nng sa sai nh hoat tnh exonuclease.
Trn ung di chuyn tng hop,nu n gp ch m nucleotide mibt cp sai, n s
lilaict b nucleotide saiv lpnucleotide ng vo.Kt qu, sau qu trnh sao
m, hai phn t DNA con uoc hnh thnh c cu tao ging ynhu phn t DNA m
ban u, m bo thng tin di truyn uoc truyn t th h ny quath h khc mt
cch chnh xc.Sao m DNA si kp dng vng ca t bo procaryoteSao m theo
kiu hnh mtS tng hop DNA dang vng vi khunDNA ca t bo procaryote
thung c dang xon kp hnh trn. Qu trnh tng hop cnguoc thuc hin t mt
im khi u (replication origine) goi l im xut pht v trin khaira c hai
pha chac ba sao m v lan dn v hai pha, cui cng tao ra hai phn t DNA
lai.C trung hop su tng hop chi xy ra v mt pha ca im khi u. Khi DNA
dang vngtrn ang sao chp, quan st thy c dang hnh con mt (eye
replication).Mt n vj sao m thng nht (t mt im xut pht) goi l
replicon.B gen ca cc sinh vt procaryote chi c mt replicon. Tc tng
hop DNA ca E. Coli cth at 50.000 nucleotide/pht, chu k sao m ko di
khong 20 phtSao m kiu hnh trn xoaySao m theo kiu hnh trn xoayNgoi
kiu sao m c dang hnh con mt nhu trn, mt s vi khun v virus cn c
kiutng hop khc goi l sao m hnh trn xoay. Qu trnh tng hop bt u bng
su ct lin ktphosphodiester tai mt im xc jnh trn mt soi DNA trn kp
tao ra hai u mt, mt ukt thc bng nhm 3OH v u kia l u 5phosphat. Su
tng hop mt mach mi mtcch lin tuc bng cch ko di mach t u 3OH ng thi
djch chuyn theo dang xoaytrn, u 5P uoc xoay ra ngoi v mt mach mi
khc uoc tng hop gin oan theookazaki, sau khi kt thc, hai phn t DNA
con uoc hnh thnhSao m t bo eucaryote t bo eucaryote c nhiu nhim sc
th, a dang, mi nhim sc th l mt phn t DNAnm lin kt vi protein, nn qu
trnh sao m phc tap. Nhiu im sao m xy ra ngthi, hay ni cch khc l
trong cng mt thi im c nhiu n vj sao m (replicon). V dunhu nm men
bnh m S. Cerevisiae c 500 replicon.Sao m t bo
eucaryoteQutrnhtnghopDNA
tboeucaryotephctaphnvtcchmhn(3.000nucleotide/pht).c im quan trong
trong qu trnh sao m t bo eucaryote l c nhiu n vj sao mxy ra, ng
thi, trn mt phn t DNA v t bo c c ch kim sot nghim ngt qutrnh ny. im
no sao qua mt ln ri th khng lp lai truc khi ton b phn t DNAuoc ti
tao hon ton.Sau khi sao m, cc DNA con c cu tao ging y nhu DNA m ban
u uoc phn chia un v mi t bo con trong qu trnh phn bo.Sa sai DNA
trong t boSa sai trong sao mSa cha nhng sai st trn DNA nhm khi phuc
lai cu trc ban u l mt c tnh camoi t bo sng. Trong qu trnh sao m, cc
baz nitt cng c th bt cp sai. Su bt cpsai thung xy ra l A bt cp sai
vi C (A...C) v G bt cp sai vi T (G...T). S d c subt cp sai nhu vy l
do trang thi tn tai ca cc baz c nhng thay i, dn n sunhn bit sai.Nh
c ch tng hop DNA lun lun khi ng t u 5' 3', nn vic sao m uoc kimsot
v sa cha mt cch chnh xc. Cc enzyme DNA-polymerase v va lm chcnng
polymer ha va c hoat tnh exonuclease theo hung 5' 3' v 3' 5'. Nu
trnung di chuyn bt gp mt nucleotide lp sai, chng s li lai ct b v lp
nucleotideng vo.Khi h thng kim tra pht hin c sai st, cc enzyme
endonuclease s ct b oan sai,sau , enzyme DNA-polymerase s tng hop
lai cho ng v enzyme DNA-ligase s nilai phuc hi trang thi bnh thung.
Khi tng hop nhn tao phn t DNA (invitro) ngui tauc tnh sai st l 105
, ngha l, trong 105 nucleotide uoc tng hop th c 1 nucleotidesai. V
du DNA trong t bo E. Coli c 3x10 nucleotide, nhu vy c sau mi ln sao
m thc 30 nucleotide sai, hay cc nhim sc th ngui c 3x10 nucleotide,
nhu vy c saumi ln sao m th c 30.000 nucleotide sai. Nu vi tn xut
sai ny th s luong t bins rt ln. Qua thuc t nghin cu cho thy tn xut
t bin trong cc qun th sinh vt lnh hn rt nhiu.Bng cch nh gi tn s t
bin xut hin trong cc qun th, ngui ta uc tnh su saist trong sao m nm
trong khong 10. Tuy nhin, trong thuc t, ti l sai st cn thp hnnhiu,
vo khong 1010 n 1011, iu ny chng t su tn tai mt h thng kim tra vsa
sai rt hiu qu ca t bo.Sa sai sau khi sao mMi trung sng v c th sng c
mi quan h mt thit vi nhau. Cc bin ng ca mitrung sng lun tc ng n cc
sinh vt. Nhng thay i ca mi trung bn ngoi ctc ng truc tip n b my di
truyn, lm bin i n.Cc tc nhn c th lm thng tn DNA trong qu trnh tn
ti- Cc tia v tru v cc tia phng xa c nng luong cao c th lm bin i cc
baz nitnhu gn thm cc nhm chc khc vo mach vng hay lm t vng, lm t cc
lin kthydro gia hai mach hay lm ct mach ca DNA,...- Cc tia cuc tm
trong nh sng mt tri thung gy nn su dimer ha cc baz thymine,hnh thnh
lin kt gia hai baz thymine nm k nhau, lm chng mt kh nng lin kt
vibaz adenine ca mach b sung.- Tc ng ca cc thnh phn trong ni bo.Su
tn thung phn t DNA c th gy ra do qu trnh trao i cht bt bnh thung gy
racc cht c hai v cc gc tu do bt loi, hay do hoat ng ca cc enzyme
khng ng blm tn ong nhng sn phm trung gian bt loi.Ngoi ra, cn c nhiu
tc nhn ha hoc khc ca mi trung bn ngoi cng gy nn subin i c th xy ra
trn DNA.Mt s kh nng gy bin i trn phn t DNA- Gy mach hay t mach: Do
soi DNA rt mnh, bn thn n lai xon cun nhiu ln nnrt d bj gy hay t
mach do tc ng bn ngoi hay khi tho xon.- Mt cc baz b sung, ngha l lm
cho baz tung ng khng c cp (nhu mt bazpurine).- Bin baz nit ny thnh
baz khc, gy nn su bt cp sai, nhu: Khi mt nhm amin,cytosine s bin n
thnh uracil hoc 5-metyl-cytosine-desamin bj nhn nhm l thymine.- Cc
baz nit c th tn tai dui 2 dang ceton v enol nn dn dn bt cp sai.- Gn
thm nhm CH3 , C2H5: Khi gn thm nhm ankyl s lm thay i tnh cht cacc
baz nit dn n bt cp sai.C ch phng nga v sa sai bo v v thch nghi vi
nhng bin i, t bo c c ch phng nga v sa sai. Ccc ch phng nga ca t bo
nhu h thng enzyme kh c, loai b cc c t, h thngiu ho cn bng cn thit
cho t bo, h thng enzyme tham gia sa sai. V du sau chothy h thng sa
sai v phng nga ca t bo- Enzyme Superoxide Dismutase (SOD) lm nhim
vu gim c: Enzyme SOD uoc phthin nm 18, c m s EC.1.15.1.1 , c mt
trong tt c cc t bo c chuyn ha oxy.Chng xc tc phn ng phn hu gc
superoxyt O2 trong t bo theo s phn ng sau2 O2 + 2H H2O2+ O2H2O2 s
uoc enzyme catalase hoc peroxydase chuyn ha. Do , SOD cng vi
catalase uoc xem l nhn t quan trong c th gii c, bo v v chng lo ha
cho t bo. Gc O2 uoc sinh ra lin tuc v cng bj phn hu khng ngng bi
hoat ng ca SOD, do , khi SOD c hoat cng cao th nng O2 cng thp.-H
thng sa sai trong t bo rt a dang v c hiu qu vi su tham gia ca cc
enzyme,nhuphotolyase, DNA-metyl-transfersase,
hthngenzymemhatronghthnggenSOS. vi khun E.Coli, do tc ng ca tia t
ngoai lm xut hin cc thymine dimer (TT) trnDNA. Khi c nh sng, enzyme
photolyase s uoc hoat ha v ct b lin kt TT, chuynv trang thi bnh
thung v n jnh.H thng enzyme nuclease c th ct b ch sai hng bng nhiu
cch v tng hop lai chong. Enzyme DNA-metyl-transferase c kh nng loai
b gc metyl trong trung hop ccbaz bj metyl ha. Tm lai, t bo c c ch
kim tra v sa nhng sai st nu c. Ngoira, t bo cn c h thng SOS (cp
cu): H thng ny hoat ng khi t bo bj tc ngmanh bi cc tc nhn gy t bin,
tao nhiu sai hng trn DNA. Trong trung hop cpbch c nhiu sai hng cn
cp cu, cc gen SOS uoc m ra. Nu sa sai khng kjp, tbo phi chp nhn hoc
bj t bin hoc cht.Trong t bo cn c h thng iu ho cn bng axit-baz, tao
iu kin thun loi cho hoatng trao i cht lm gim kh nng tao cc cht c
hai. sinh vt a bo, vic kh c v loai b nhiu ha cht gy c uoc m bo bi
ganv thn.
