“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic AP Biology material. ” Watson & Crick
Directionality of DNA § You need to PO4
nucleotide
number the carbons! u
it matters!
N base 5ʹ CH2
This will be IMPORTANT!!
4ʹ
O
3ʹ AP Biology
1ʹ
ribose
OH
2ʹ
The DNA backbone § Putting the DNA backbone together u
refer to the 3ʹ and 5ʹ ends of the DNA § the last trailing carbon Sounds trivial, but… this will be IMPORTANT!!
5ʹ PO4 5ʹ CH2 4ʹ
base O 1ʹ
C 3ʹ
O –O P O O 5ʹ CH 2
2ʹ
base O
4ʹ
1ʹ 2ʹ
3ʹ
OH
AP Biology
3ʹ
Anti-parallel strands § Nucleotides in DNA backbone are bonded from phosphate to sugar between 3ʹ & 5ʹ carbons
5ʹ
3ʹ
3ʹ
5ʹ
DNA molecule has “direction” u complementary strand runs in opposite direction u
AP Biology
Bonding in DNA 5ʹ
hydrogen bonds
3ʹ
covalent phosphodiester bonds
3ʹ
5ʹ
….strong or weak bonds? AP Biology How do the bonds fit the mechanism for copying DNA?
Base pairing in DNA § Purines adenine (A) u guanine (G) u
§ Pyrimidines thymine (T) u cytosine (C) u
§ Pairing u
A:T § 2 bonds
u AP Biology
C:G § 3 bonds
Copying DNA § Replication of DNA base pairing allows each strand to serve as a template for a new strand u new strand is 1/2 parent template & 1/2 new DNA u
AP Biology
DNA Replication
Let’s meet the team…
§ Large team of enzymes coordinates replication
AP Biology
Replication: 1st step § Unwind DNA u
helicase enzyme § unwinds part of DNA helix § stabilized by single-stranded binding proteins helicase
single-stranded binding proteins AP Biology
replication fork
Replication: 2nd step § Build daughter DNA strand add new complementary bases u DNA polymerase III u
DNA Polymerase III AP Biology
But… Where’s the We’re missing ENERGY something! for the bonding! What?
Energy of Replication Where does energy for bonding usually come from? We come with our own energy!
You remember ATP! Are there there Are otherenergy ways other to get energy nucleotides? out You of betit? !
ATP GTP CTP TTP AP Biology
modified nucleotide
And we leave behind a nucleotide!
energy energy
CMP TMP GMP AMP ADP
Energy of Replication § The nucleotides arrive as nucleosides u
DNA bases with P–P–P § P-P-P = energy for bonding
DNA bases arrive with their own energy source for bonding u bonded by enzyme: DNA polymerase III u
ATP AP Biology
GTP
TTP
CTP
5ʹ
Replication § Adding bases u
can only add nucleotides to 3ʹ end of a growing DNA strand § need a “starter”
nucleotide to bond to u
strand only grows 5ʹ→3ʹ
AP Biology
B.Y.O. ENERGY! The energy rules the process
3ʹ
energy
DNA Polymerase III
energy
DNA Polymerase III
energy
DNA Polymerase III DNA Polymerase III
energy
3ʹ
5ʹ
5ʹ
3ʹ
5ʹ
need “primer” bases to add on to
3ʹ
energy no energy to bond
û
energy energy
energy energy
ligase energy
energy
3ʹ
AP Biology
5ʹ
3ʹ
5ʹ
Okazaki
Leading & Lagging strands Limits of DNA polymerase III u
can only build onto 3ʹ end of an existing DNA strand Ok
ragm f i k a z a 3ʹ
5ʹ
3ʹ
5ʹ
5ʹ
ents 3ʹ
5ʹ
ligase
growing 3ʹ replication fork
5ʹ
5ʹ
Lagging strand
Leading strand 3ʹ
Lagging strand Okazaki fragments u joined by ligase u
AP Biology § “spot
welder” enzyme
û
3ʹ
!
5ʹ 3ʹ
DNA polymerase III
Leading strand u
continuous synthesis
Replication fork / Replication bubble 3ʹ
5ʹ
5ʹ
3ʹ
DNA polymerase III
leading strand 5ʹ
3ʹ 5ʹ
3ʹ
3ʹ 5ʹ
5ʹ
5ʹ
3ʹ
lagging strand
3ʹ 5ʹ
3ʹ 5ʹ lagging strand
5ʹ
5ʹ leading strand 3ʹ
growing replication fork 3ʹ
leading strand
lagging strand 5ʹ 5ʹ
AP Biology
growing replication fork 5ʹ
5ʹ
5ʹ
3ʹ
Starting DNA synthesis: RNA primers Limits of DNA polymerase III u
can only build onto 3ʹ end of an existing DNA strand
5ʹ
3ʹ 3ʹ
5ʹ
5ʹ
3ʹ
5ʹ
3ʹ
5ʹ growing 3ʹ replication fork
DNA polymerase III
primase RNA 5ʹ
RNA primer built by primase u serves as starter sequence DNA polymerase III AP for Biology u
3ʹ
Replacing RNA primers with DNA DNA polymerase I u
removes sections of RNA primer and replaces with DNA nucleotides
3ʹ
5ʹ
DNA polymerase I
5ʹ
5ʹ 3ʹ
ligase
growing 3ʹ replication fork RNA
5ʹ 3ʹ
But DNA polymerase I still can only build onto 3ʹ end of an existing DNA strand AP Biology
Chromosome erosion All DNA polymerases can only add to 3ʹ end of an existing DNA strand
Houston, we have a problem!
DNA polymerase I 5ʹ 3ʹ
3ʹ
5ʹ
5ʹ growing 3ʹ replication fork
DNA polymerase III RNA
Loss of bases at 5ʹ ends in every replication chromosomes get shorter with each replication AP Biologyto number of cell divisions? u limit u
5ʹ 3ʹ
Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap u
5ʹ
limit to ~50 cell divisions
3ʹ 3ʹ
5ʹ
5ʹ growing 3ʹ replication fork
telomerase
Telomerase TTAAGGG TTAAGGG TTAAGGG enzyme extends telomeres u can add DNA bases at 5ʹ end u different level of activity in different cells
u
AP Biology § high
in stem cells & cancers -- Why?
5ʹ 3ʹ
Replication fork DNA polymerase I
5’ 3’
DNA polymerase III
ligase
lagging strand primase
Okazaki fragments
5’
SSB
3’
5’ 3’
3’ 5’ helicase
DNA polymerase III
leading strand direction of replication
AP Biology
SSB = single-stranded binding proteins
DNA polymerases § DNA polymerase III 1000 bases/second! u main DNA builder u
Roger Kornberg 2006
§ DNA polymerase I 20 bases/second u editing, repair & primer removal u
DNA polymerase III enzyme
AP Biology
Arthur Kornberg 1959
Editing & proofreading DNA § 1000 bases/second = lots of typos!
§ DNA polymerase I u
proofreads & corrects typos
u
repairs mismatched bases
u
removes abnormal bases § repairs damage
throughout life u
AP Biology
reduces error rate from 1 in 10,000 to 1 in 100 million bases