1 10/25/2015 DNA l Filename DNA.ppt l Developed by D.Thomas l Updated Oct 1999 by H.B. Fackrell Material for Introductory Microbiology

2 10/25/2015 The quick brown fox jumps over the lazy dog Deoxy- ribo- nucleic acid

3 10/25/2015

4 10/25/2015 O Base C P Pentose Nucleoside O Base C Nucleo t ide

5 10/25/2015 CH 2 NH 2 N OH N N N Adenine Ribose O-P-O O O Phospha t e 5' The nucleo t ide, adenosine 5’ monophosphate (AMP) Nucleotide

6 10/25/2015 CH 2 NH 2 N OH O N N N Adenine Ribose Cousins of AMP The energetic.... Adenosine triphosphate (ATP) O-P-O O O O-P- O O O O (Yet another nucleo t ide)

7 10/25/2015 The event The event of 20th Century biology

8 10/25/2015 The event of 20thC biology l A paper published in Nature 171: l Not a lengthy paper, but it contains the words that started a revolution in biology l Dateline 1953 l Authors, J.D. Watson and F.H.C. Crick l Title: A structure for deoxyribose nucleic acid

9 10/25/2015 DNA strands are complementary POHPPP P PP P 5’ 3’ CCAT A T G G WATSON KCIRC

10 10/25/2015 Base* P - CH2 1' 2' 3' 4' O OHH 2’ deoxynucleoside monophosphate *A, T, G or C Sugar

11 10/25/2015 POHPPP P PP P 5’ 3’ CCAT A TG G PHOSPHODIESTER BONDS

12 10/25/2015 Base 1 Base 2 (sugar)- 3’OH HO-P-O-(sugar) + Base 1 (sugar)- -(sugar) O O O-P-O + H2OH2O O Phosphodiester bond O Base 2 -3’OH

13 10/25/2015 P OH PP P P PP P 5’ 3’ CCAT A T G G Complementary strands interact by hydrogen bonding of specific base pairs A=C G=C

14 10/25/2015 NN N H H H C H 1’ CH 3 O H O N H N O N C NH H H Base pairing Adenine Thymine Guanine Cytosine 1’ 1’ 1’ refers to the 1’ carbon of deoxyribose

15 10/25/2015 Purine Pyrimidine NN N H H H C H 1’ CH 3 O Hydrogen bonding A T = 1’

16 10/25/2015 H O N H N O N C NH H H Hydrogen bonding G C 1’ 1’ Purine Pyrimidine

17 10/25/2015

18 10/25/2015 n Each strand has a free 5’ end and a free 3’ end n Strands are antiparallel P OH PP P P PP P 5’ 3’ CCAT A T G G

19 10/25/2015 n Strands hydrogen bonded by specific base pairing (A loves T; G C!) n Strands are complementary POHPPP P PP P 5’ 3’ CCAT A T G G

20 10/25/2015 Base Deoxyribose (sugar) 1' 2' 3' 4' 5' P = O O O OP c5 P P P OH Base Sugar Phosphate

21 10/25/2015 Base 1' 2' 3' 4' 5' c5 P P P OH Bases form the “rungs” of the DNA ladder

22 10/25/2015 1' 2' 3' 4' 5' c5 P P P OH Sugar-phosphates form each “upright” of the DNA ladder Sugar S S S S S P S P P

23 10/25/2015 1' 2' 3' 4' 5' c5 C5 P P P OH 5’ end 3’end The 5’ end and the 3’end are different the 3’end are different

24 10/25/2015 DNA replication requires l DNA template l Primer on which to add new nucleotides l DNA polymerase (enzyme) l Nucleotides –deoxy adenosine triphosphate (dATP) –dGTP –dCTP –dTTP l ATP, Mg ++

25 10/25/2015 DNA replication Template determines which nucleotide to add next 3’OH Primer determines where the next nucleotide is added The next nucleotide

26 10/25/2015 Base* P - CH2 1' 2' 3' 4' O OHH 3’-OH where the next nucleotide will be added

27 10/25/2015 Growth of a nucleic acid POHPP PPP 5’ 3’ B1B1 B3B3 B2B2 B4B4 OH 3’ B5B5 PPP 5’ PPP PPP 5’ 3’ B1B1 B3B3 B2B2 B4B4 OH 3’ B5B5 + PP 5’ P Growth is from 5’ to 3’ end

28 10/25/2015 POHPPP P P P 5’ 3’ CC A C TG G OH Template determines what the next base will be (A) Primer 3’OH is where the next base will go Next base

29 10/25/2015 Replication “eye” or “bubble” Double-stranded (ds DNA) Origin of replication

30 10/25/2015 DNA replication Replication “fork” in one corner of “eye”

31 10/25/2015 DNA replication Parent strands Daughter strands

32 10/25/2015 DNA replication 5’ 3’ 5’ 3’ 5’ 3’

33 10/25/2015 <- As the eye enlarges... 5’ 3’ 5’ 3’ 5’ 3’ 5’ This strand, the leading strand, can elongate freely. DNA synthesis is continuous towards corner of “eye” This strand, the lagging strand, grows as fragments which are later ligated together 3’

34 10/25/2015 Okazaki fragments (DNA) 5’ 3’ 5’ 3’ 5’ 3’ 5’ Leading strand (continuous strand) Lagging or discontinuous strand Fragments, before they ligate together are named after their discoverer, Okazaki 3’

35 10/25/2015 Replication involves many origins

36 10/25/2015 Replication of DNA needs several enzymes l Helicase (to unwind the alpha helix) l Primase (to build RNA primer segments) l DNA polymerase (to extend primers, and more) l DNA ligase (to link together the ends of adjacent DNA fragments

37 10/25/2015 Replication of DNA in brief (5 steps) (5 steps) ss DNA RNA primer 1) synthesis of RNA primer (evolutionary implications?)

38 10/25/2015 Replication of DNA in brief (5 steps) (5 steps) ss DNA (old) RNA primer 2) added to RNA primer by DNA 2) New DNA added to RNA primer by DNA polymerase polymerase 3’5’ 3’5’

39 10/25/2015 Replication of DNA in brief (5 steps) (5 steps) ss DNA (old) 3) RNA primer cleaved out (by DNA polymerase) 3’ 5’

40 10/25/2015 Replication of DNA in brief (5 steps) (5 steps) ss DNA (old) 4) synthesized (DNA polymerase) where RNA primer cleaved out 4) New DNA synthesized (DNA polymerase) where RNA primer cleaved out 3’ 5’

41 10/25/2015 Replication of DNA in brief (5 steps) (5 steps) ss DNA (old) 5) 5) DNA ligase joins 3’ end of each newly completed fragment with 5’ end of the next to give: 3’ 5’ 3’ 5’ parent strand new complementary strand 3’5’

42 10/25/2015 POH A A’ Given, DNA, single stranded in the middle, ds at ends 1. For the B segment, which end, B or B’ represents the 3’ end? BB’ 2.. For the A fragment, which end, A or A’ represents the 3’ end? 3.. If we fill the gap in a test tube (in vitro) reaction containing only NTP’s and DNA polymerase, how many pieces will the lower strand contain?

43 10/25/2015 In the lab, DNA strands can be separated and re-annealed Heat, alkali Cool, neutral pH

44 10/25/2015 DNA strands can be separated and re- annealed Amazing/ It must be our destiny! our destiny! “By Jove, if it isn’t....”

45 10/25/2015 DNA strands can be separated and re-annealed I mportant in nature I mportant in nature I mportant in biological sciences I mportant in biological sciences

46 10/25/2015 The qui C k brown fox jumps over T he l A zy do G