Genetics: Replication

Front 1

the basics of replication

Back 1

-DNA replicates in a semi-conservative fashion and is composed to two anti-parallel strands
-DNA replication USUALLY proceeds bidirectionally, but it can proceed in one direction too
-bidirectional replication implies that there are TWO replication forks (going in opposite direction) for every origin of replication

Front 2

What direction does DNA synthesis occur in? What direction is DNA read in?

Back 2

DNA is synthesized 5' --> 3'

the DNA template is read 3' --> 5'

Front 3

how is a strand of DNA lengthened?

Back 3

the incoming nucleotide, a deoxynucleoside (dNTP), will lose two phosphates and attach via Hydrogen Bond* linkage to the opposing nucleotide on the template strand (i.e. A pairing with T), and via a Phosphodiester Linkage* to the adjacent nucleotide on the growing DNA strand that it adds onto

USING DNA POLYMERASE

Front 4

Transition state during DNA elongation

Back 4

DNA polymerase will form a transition state involving THREE Aspartate residues and TWO Mg2+ ions

the Aspartates are coordinated with Mg2+ which functions to:
1. prepare the attack of 3'OH of the growing DNA chain on the alpha phosphate of the incoming dNTP, and
2. to stabilize the beta- and gamma-phosphates, which will be irreversible broken down into two inorganic phosphates

Front 5

what do DNA Polymerases require?

Back 5

both a TEMPLATE to read off of, and a PRIMER to get started

Front 6

accuracy of DNA Polymerase

Back 6

the rate of mutation is super low
-DNA Polymerase MUST be accurate
-has Proofreading ability
-only A bound to T, or C bound to G, will fit perfectly into the active site of the polymerase. Any other mismatched pair will signal that somethings wrong.

-also has editing function (exonuclease activity)

Front 7

exonuclease activity of DNA polymerase I

Back 7

new DNA is synthesized from 5' -->3'

as soon as a mismatch arises, the DNA polymerase will backtrack in the 3' --> 5' direction to remove the mismatched nucleotide, and corrects it with the right one and will move forward again in the 5' --> 3' direction

thus, DNA polymerase is capable of moving in both 5' --> 3' and 3' --> 5' directions

Front 8

distinguishing the different classes of DNA polymerases in E. coli

Back 8

ALL have 3' --> 5' Exonuclease (proofreading) capabilities

DNA Pol I is the only one with 5' --> 3' Exonuclease activity

DNA Pol III has the fastest rate of DNA synthesis

Front 9

Structure of DNA Polymerase III

Back 9

THREE beta-clamps (dimer)
ONE clamp loader (pentamer)
TWO core units (which carry out the actual DNA polymerase activity)

(Note: Helicase is NOT part of the DNA polymerase, but it does interact with it later on to unwind DNA)

two beta subunits form the beta clamp

Front 10

Replication: Initiation

Back 10

The Origin of Replication is composed of two parts:
1. DNA Unwinding Element (DUE)** A section rich in Adenine-Thymine pairs
2. EIGHT binding sites for DnaA protein

Steps:
1. DnaA, with the help of ATP, binds to ALL eight binding sites, creating a torsional strain
2. this strain will cause DNA to be denatured in the DUE region, so the strand separates
3. DnaB (aka HELICASE) binds to the newly opened DUE region with the help of DnaC-ATP
4. since helicase is docked and ready to do its thing, replication begins

Front 11

DUE

Back 11

DNA Unwinding Element

three 13-bp sequences in a row at the start of the replication origin

AT-rich region: this is important because for replication to occur you need to separate the strands. AT only have 2 hydrogen bonds, whereas CG have 3. So its easier to denature and separate AT sequences and be able to start replication.

Front 12

DNA Gyrase ("DNA Topoisomerase II")

Back 12

when the DnaB helicase starts to unwind the DNA, it creates a torsional stress on the DNA strand.
DNA Gyrase relieves this stress

Front 13

primase

Back 13

located next to the helicase

synthesizes a short RNA primer to serve as a starting point for replication (this RNA primer will need to be replaced because we ultimately want DNA, not RNA)

Front 14

single stranded binding proteins (SSB)

Back 14

stabilize the single-stranded fragments that have not replicated yet

without this, the single-stranded DNA will be targeted for degradation

Front 15

what happens after the RNA primer is laid down?

Back 15

DNA POlymerase III* comes in and starts adding on nucleotides to the primer to form Okazaki Fragments

nucleotides will be added on until it meets the RNA primer that was part of the PREVIOUS Okazaki fragment

at this point, the primes will make another RNA primer, and the cycle continues.

Front 16

DNA Polymerase and the Lagging Strand

Back 16

DNA polymerase will interact/bind with the helicase, and move together in the direction of the opening replication fork

the lagging strand is looped around and then fed into the Beta-Clamp and core

Front 17

DNA Polymerase beta-clamps

Back 17

there are 3 beta-clamps on ONE DNA Polymerase Complex

-the beta-clamp associated with the leading strand will always stay there
-the beta-clamp in the middle that is in the clamp-loading pentameric subunit will essentially receive each new primer that is made, and ultimately slide over to the lagging strand's beta-clamp positions and clamp down on the DNA so it can continue adding nucleotides
-the old beta-clamp gets discarded

Front 18

how do we replace the okazaki fragments?

Back 18

at some point, the RNA primers of the okazaki fragments will need to be replaced by the exonuclease function of the DNA Polymerase I*
-this exonuclease activity is a bit different from the 3' --> 5' exonuclease (which functions in proofreading and removal of mismatched base pairs) in that it removes nucleotides from the RNA primer and replaces them with DNA nucleotides in the 5' --> 3' direction***

Front 19

The DNA Polymerase III Clamp Loader

Back 19

there are 5 subunits of the clamp-loading complex

the complex binds 3 ATP, and also binds to a dimeric beta-clamp

the binding to the beta clamp forces the beta-clamp to open at one of its two subunit interfaces, allowing it to interact with the DNA

hydrolysis of the bound ATP allows the beta-clamp to close again around the DNA

Front 20

DNA Ligase

Back 20

seals fragments with the help of ATP

creates phosphodiester bonds between the 2 nucleotides

Front 21

Replication Termination

Back 21

In the case of E. coli DNA (circular chromosome), the bidirectionally of an origin of replication means that the two replication forks will eventually meet each other

when that happens, there will be Termination Sequences (Ter) that are bound by Terminus Utilization Sequences (Tus). WHen this complex forms, a so-called "polymerase trap" occurs, and you'll end up with "Catenated" Chromosomes (linked together)

then DNA Topoisomerase IV comes in, and basically cuts one of the DNA strands so that the other replicated DNA strand can detach, ultimately forming two independent chromosomes

Front 22

Topoisomerase IV

Back 22

. Replication of the DNA separating opposing replication forks leaves the completed chromosomes joined as catenanes, or topologically interlinked circles. The circles are not covalently linked, but because they are interwound and each is covalently closed, they cannot be separated—except by the action of topoisomerases

comes in during termination and targets Catenated chromosomes in prokaryotes

cuts one of the DNA strands so that the other replicated DNA strand can detach, ultimately forming two independent chromosomes

Front 23

Eukaryotic Replication

Back 23

same mechanisms, but some differences:
1. There are multiple Origins of Replication
2. The Origin Recognition Complex found in euks is analogous to the DnaA that binds to the specific origin replication sites. Both DnaA and ORC will require ATP
3. The Minichromosome Maintenance Protein (MCM) is analogous to the DnaB helicase found in proks
4. Together, the ORC and MCM make up the Pre-Replicative Complex. Once this complex forms, it becomes LICENSED.
5. DNA Polymerase delta is analogous to the bacterial DNA Polymerase III

Front 24

Assembly of a Pre-Replicative Complex at a Eukaryotic Replication Origin

Back 24

**key step in the initiation of replication in eukaryotes**

(analogous to the loading of the bacterial DnaB helicase by DnaC protein)

loading of the MCM helicase complex onto the DNA forms the pre-replicative complex (pre-RC)

once this complex forms it becomes LICENSED, which basically means its all ready to go if it passes ONE last checkpoint that is dependent on cyclin-dependent CDK (Cell Division Kinases)***

Front 25

Eukaryotic DNA Polymreases

Back 25

DNA Polymerase delta is analogous to the bacterial DNA Polymerase III

the DNA Polymerase delta will be most prevalent in the S phase of the cell cycle, when DNA replication occurs

Front 26

why is regulation so important in the initiation step of eukaryotes?

Back 26

for eukaryotes especially, you want to make sure that replication occurs only ONCE in each cell cycle

Front 27

Chromatin

Back 27

more condensed chromatin will replicate later in the S phase**
(at the 6-8 hour mark)

compared to less dense chromatin that is replicated first (0-2 hour mark)

Front 28

telomeres

Back 28

telomeres are nucleotide repeat sequences at the tips of the chromosome

they serve to prevent the DNA from having a recognizable "end", and thats why theres a little LOOPED structure where the DNA folds back onto itself so that the DNA can still be continuous and not just end abruptly at the tip

**the enzyme Telomerase that catalyzes the addition of nucleotides uses an RNA (not DNA!!!!) template* in order to synthesize the growing telomere strand of DNA

telomeres shorten naturally with constant cell divisions and ultimately with age, so telomerase tries to help out to extend the ends of the chromosome