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Molecular Biology IIDr. Bipinraj N K

09373303308bipinrajnk@gmail.com

mailto:bipinrajnk@gmail.commailto:bipinrajnk@gmail.com

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What is molecular biology ? The attempt to understand biological phenomena in molecular termsOr The study of gene structure and function at the molecular level

Di erent biological phenomena ? Origin of life: Inheritance:

Mendels or! "hromosome Theory of Inheritance #ecombination and gene mapping Discovery of D$% #elation bet een gene and proteins Mutation

Molecular nature of gene $ature of genetic material "hemical physical

characteristics &tructure D$%

'ene function 'ene to proteins #eplication 'ene regulation s

%pplications #ecombinant technology "loning Molecular tools

What is molecular biology ?

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La o! Dominance" #ome allele$ are %ominan& 'ileo&'er$ are rece$$i(e) an organi$m i&' a& lea$& one%ominan& allele ill %i$play &'e e*ec& o! &'e %ominan&allele

La o! #egrega&ion" During game&e !orma&ion+ &'e

allele$ !or eac' gene $egrega&e !rom eac' o&'er $o &'a&eac' game&e carrie$ only one allele !or eac' gene. La o! In%epen%en& ,$$or&men&" -ene$ !or %i*eren&

&rai&$ can $egrega&e in%epen%en&ly %uring &'e !orma&io! game&e$.

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D$% #eplication 'eneral (eatures of D$% #eplication

&emiconservative &emi)discontinuous #eplication

#e*uires a primer

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,ccor%ing &o &'e a&$on/ rick mo%el &'ere are &'reepo$$ibili&ie$

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In 1928+ Ma&&'e Meselson an% ranklin &tahl per!orcla$$ic e4perimen& &o %i$&ingui$' among &'e$e &'reepo$$ibili&ie$. 5'ey labele% E. coli DN, i&' 'ea(y ni&rogen 6 12 N by

gro ing cell$ in a me%ium enric'e% in &'i$ ni&rogen i$o&ope.

5'i$ ma%e &'e DN, %en$er &'an normal 5'en &'ey $ i&c'e% &'e cell$ &o an or%inary me%ium

con&aining primarily 1 N+ !or (ariou$ leng&'$ o! &ime. inally+ &'ey $ubjec&e% &'e DN, &o "s"l gradient

ultracentrifugation &o %e&ermine &'e %en$i&y o! &'e D

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DN, eplica&ion D$% replication i$ &'e proce$$ o! pro%ucing & o i%en&ical replica$ !rom one original DN,

molecule Ini&ia&ion+ elonga&ion an% &ermina&ion Ini&ia&ion"

Ini&ia&ion" Ini&ia&ion $&ar&$ a& cer&ain poin&$ calle% a$ origin$ con&rolle% by primo$ome$: origin recocomple4. 6uni& o! replica&ion i$ replicon

;rimo$ome con$i$&$ o! (ariou$ en

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Ini&ia&ion ;riming in = oli ;rimo$ome " omple4 o! pro&ein$ in(ol(e% in priming or

ini&ia&ion o! replica&ion. I& i$ a mobile uni& i& al$o $yn&'e$i$prima$e !or replica&ionImportant proteins+

en,ymes(unctions

Dna % Bin%$ &o 9/mer uni&$-. i$&one like pro&ein$ $&abili

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Important sites (unction

>ri 'ere replica&ion $&ar&$

9/mer 6in$i%e &'e >ri Dna , bin%$ &o ini&ia&e mel&ingor opening

13/mer Le!& &o 9/mer 'ere Dna Bbin%$ an% open$ &'e %ouble'eli4

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In eukaryo&e$ origine o! replica&ion i$

con&rolle% by a origin recogni&ion com6>> bin%$ &o an origin& o !ac&or$+ cell %i(i$ion cycle A 6 Dan% D51+ are recrui&e% an% 'a(e &'e ro! loa%ing &'e minic'romo$omemain&enance pro&ein 6M M comple4M M C7 on&o &'e replica&ion origin

5'e M M C7 comple4 i$ a 'e&ero'e4amer&'a& 'a$ ,5;a$e/%epen%en& DN, 'elica$ac&i(i&y an% !orm$ a ring aroun% &'e a& replica&ion origin$,!&er &'i$ 'elica$e 'a$ boun% DN,+ &'eorigin$ are licen$e%+ marking &'e en% pre/replica&ion comple4 6pre a$$embly

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DN, ;olymera$e$

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;olymera$e I 6;ol I " Di$co(ere% by ,r&'ur Kornberg in 192A an% on Nobel

pri

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;ol III

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DN, %amage an% repair

DN, Damage" DN, can be %amage% in many %i*eren& ay$+ an% &'i$%amage+ i! le!& unrepaire%+ can lea% &o mu&a&ion$.

DNA damage is not the same as mutation, although it canlead to mutation

Damage "aused by %l!ylation of /ases

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Damage aused by %l!ylation of /ases ,lkyla&ion i$ &'e a%%i&ion o! alkyl group &o &'e

nega&i(ely c'arge% group$ Impor&an& alkyla&ion $i&e$ are

N7 o! guanine " generally 'armle$$ N3 o! a%enine " crea&e$ noncoding base : cytoto4ic

>A o! guanine " ,lkyla&ion on >A c'ange$ &'e&au&omeric !orm o! guanine $o i& ba$e/pair$ na&urallyi&' &'ymine an% cau$ing &'e mu&a&ion in &'e ne4&replica&ion.

=g. Me&'ylme&'ane $ul!ona&e 6MM# =&'ylni&ro$ourea 6=NH =&'ylme&'ane $ul!ona&e 6=M#

Damage "aused by .ltraviolet

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g y#adiation

.ltraviolet 0.21 radiation cro$$/link$a%jacen& pyrimi%ine$ on &'e $ame DN, $&ran%+!orming & o major le$ion$

"yclobutane pyrimidine dimers 6"5Ds "

cycli

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DN epair " Direc& epair

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DN, epair Direc& epair ;'o&oreac&i(a&ion 6,lber& Kelner 19 0 "

5'ere are & o en

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=4ci$ion epair /ase e4cision repair 0/ #1 D$% glycosylase detects the damaged

base an% break$ i&$ glyco$i%ic bon% i&' &'e$ugar crea&ing an apurinic or apyrimidinicsite 0%5 site1;

%5 endonuclease cu&$+ or nic!s; &'e DN,$&ran% on &'e 2 /$i%e o! &'e ,; $i&e.

DN, p'o$p'o%ie$&era$e remo(e$ &'e ,; $ugarp'o$p'a&e

DN, polymera$e I per!orm$ repair $yn&'e$i$ by%egra%ing DN, in &'e 2 E3 %irec&ion 'ileFlling in i&' ne DN,

DN, liga$e $eal$ &'e remaining nick &ocomple&e &'e repair

6b glyco$yla$e remo(e$ &'e

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6b glyco$yla$e remo(e$ & e6c ,;=1 purinic:apyrimi%inicen%onuclea$e clea(e$ on &'&'e apyrimi%inic $i&e6% DN, polymera$e correc&'e gap6e DN, liga$e I $eal$ &'e nicre&urning &'e DN, &o norm0f1 >cca$ionally+ &'e DN, pmake$ a mi$&ake.0g1 ,;=1 u$e$ i&$ 39/e4onuremo(e &'e mi$paire% 5+ aglea(ing a gap0h1 5'i$ &ime+ DN, polymecorrec&ly place$ a 6blue ac&'e -.

$ucleotide 4cision #epair

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p The e4cision en,yme u(r,B

en%onuclea$e 63 polypep&i%e cu&$&'e %amage% DN,+ pro%ucing anoligonucleo&i%e &'a& i$ 1 C13 ba$e$long in prokaryo&e an% C3 n& ineukaryo&e

Global Genome NER

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; + &oge&'er i&' ano&'er pro&ein calle%' 3B+ recogni

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5ran$crip&ion couple% N= 5ran$crip&ion/couple% N= u$e$ all o! &'e $ame !ac&or$ a$ %oe$ global genome

N= + e4cep& !or ; . ecogni&ion o! &'e %amage in 5 N= i$ %one by N, polymera$e 'en N, polymera$e encoun&er$ a %i$&or&ion o! &'e %ouble 'eli4 cau$e% by

DN, %amage+ i& $&all$. 5'i$ place$ &'e bubble o! mel&e% DN,+ 'ic' i$ crea&e% by &'e polymera$e+ a&

&'e $i&e o! &'e le$ion ,& &'a& poin&+ ;, coul% recogni

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Double)&trand /rea! #epair in u!aryotes: Double $&ran%e% break$ are broken c'romo$ome$+ i! i& i$ no&

repaire% i& can lea% &o cell %ea&'+ c'romo$omal aberra&ion+mu&a&ion e&c.

=ukaryo&ic cell 'an%le &'e break$ in & o ay$ omologou$ recombina&ion Non/'omologou$ en% joining

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