DNA Replication 2007-2008.

DNA Replication

Before we do ANYTHING else…
WHY is DNA important? Why, because it makes up the genetic material, Mrs. Coleman! OK, but WHY is the GENETIC MATERIAL important? Why, because it does two important things: The genetic material in each living cell tells that cell exactly HOW to CREATE ITSELF, AND HOW to FUNCTION The genetic material also allows these instructions to be passed onto to every offspring generation

Monomers = Nucleotides
DNA Structure DNA is a NUCLEIC ACID Nucleic Acids are POLYMERS The MONOMERS that make up Nucleic Acid polymers are NUCLEOTIDES. Polymer = Nucleic Acid Monomers = Nucleotides

Nucleotide Structure P S BASE Each nucleotide is composed of 3 parts
Sugar Nitrogenous base Phosphate group Of the 3 nucleotide parts, ONLY the nitrogenous base may vary. The phosphate and sugar are always the same. P S BASE

The Bases There are two families of nitrogenous bases
Purines are BIGGER (two rings) than Pyrimidines (one ring) Pyrimidines Cytosine Thymine Uracil Purines Adenine Guanine

The Sugar 5 carbon sugar (also known as a pentose sugar)
In DNA the sugar is deoxyribose In RNA the sugar is ribose There is an oxygen atom clearly marked in deoxyribose The carbons are indicated at the bends in the ring and are numbered to indicate position. This is important!

The Phosphate Group The phosphate group is always attached to the 5’ carbon

Linking Nucleotides together to make DNA
Covalent bonds link one nucleotide to the next The type of covalent bond involved is called a phosphodiester bond These bonds are formed between the sugar of one nucleotide and the phosphate group of the next nucleotide in the chain

Linking Nucleotides together to make DNA
The sugar and phosphate molecules make the backbone of any nucleic acid The bases may vary, but the backbone of “sugar-phosphate-sugar-phosphate” remains the same

Double Helix The DNA molecule is composed of two “polynucleotide” molecules that spiral around an imaginary axis. This is known as a “double helix”. Click here for another DNA model site.

Double Helix Structure
Sugar/phosphate backbones are on the outside of the helix Nitrogenous bases are paired in the inside of the helix Hydrogen bonds between bases hold the two strands of DNA together

Bonding in DNA 5 3 3 5 hydrogen bonds covalent phosphodiester
….strong or weak bonds? How do the bonds fit the mechanism for copying DNA?

Only certain bases are compatible with each other Purine + Pyrimidine Cytosine + Guanine Adenine + Thymine

Complementarity The two strands of the double helix are said to be complementary. A - T C - G This means that each is a predictable counterpart of the other.

Discovering the Structure of DNA
Once it was determined that DNA was the genetic material, the next step was to determine the structure of the molecule It was hoped once the structure was determined, clues would be evident about how the molecule worked.

Double helix structure of DNA
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

Scientists Working on the DNA Molecule in the 1950s
Linus Pauling Very well known scientist Made many important discoveries in molecular biology Especially important discoveries about proteins Discovered the alpha helix shape of some protein molecules Still, he ultimately “lost” the race in determining the structure of DNA

Rosalind Franklin Working in the lab of Maurice Wilkins at King’s College (in England) Made excellent X-ray crystallography images of the DNA molecule that were ultimately used to discover DNA’s structure

Photo 51 The x-ray diffraction image made by Franklin and shown to Watson by Maurice Wilkins

Maurice Wilkins It was Wilkins’ lab in which Rosalind Franklin came to work at King’s College Was, along with Watson and Crick, awarded the Nobel Prize.

James Watson Very young American Came to study at the Cavendish in England Interested in DNA Using Rosalind Franklin’s DNA images, he and Francis Crick determined DNA’s structure and were awarded the Nobel Prize.

Francis Crick British Ph.D student at at the Cavendish Laboratory in England. Shared a common interest with James Watson in DNA Using Rosalind Franklin’s DNA images, he and Watson determined DNA’s structure and were awarded the Nobel Prize

Puzzle of DNA Structure
Initially, Watson tried to make the bases fit together “like with like” Purine + purine was too wide and pyrimidine + pyrimidine was too narrow – the structure simply could not fit together. Eventually he realized that purine and pyrimidine would fit together where like with like would not.

Puzzle of DNA Structure
Figuring out the A-T and C-G arrangement of DNA also explained “Chargaff’s Rules”. That A’s and T’s occurred in a 1:1 ratio as did C’s and G’s.

DNA Structure Though the rungs of the DNA ladder must always be A-T and C-G, the SEQUENCE of these bases along the length of the strand is NOT restricted. Thus the sequence can be varied in many ways, allowing for nearly unlimited variety among organisms.

Announcement of the Structure of DNA
Watson and Crick announced their findings in the April 1953 issue of the journal “Nature”. Their article was only one page long. The beauty of the model that Watson and Crick described was that it immediately suggested a mechanism by which DNA could replicate itself

1953 article in Nature Watson and Crick

2nd Big Question: How is DNA actually packaged inside a cell?
Remember: You have a LOT of DNA packed into the nucleus of each cell in your body. If you took out all the DNA in ONE cell and stretched it out, it would be 3 meters long!! How can you get all this stuff in such a small space?

Organizing DNA DNA DNA is organized into chromosomes histones
ACTGGTCAGGCAATGTC DNA Organizing DNA DNA is organized into chromosomes double helix DNA molecule wrapped around histone proteins like thread on spools Then these “spools” are FURTHER coiled around each other! DNA-protein complex = chromatin All these coils are organized into a single long thin fiber A CHROMOSOME Remember that before a cell can divide to make more cells, it must COPY the genetic material Chromosomes must be duplicated exactly Then these DUPLICATED Chromosmes get condensed even further during cell division to look like the familiar textbook drawings of double stranded chromsomes we’re used to seeing. histones chromatin Condensed, double stranded chromosome

3rd Big question… DNA REPLICATION
How exactly does this “duplication” of the genetic material happen? On the surface it’s pretty simple, but we’re gonna learn some more detail… No, don’t thank me… DNA REPLICATION

DNA Replication During replication, the pairing of the bases enables existing DNA strands to serve as templates for new, complementary strands.

DNA Replication The first step in replication is the separation of the strands

DNA Replication The second step in replication
Each “old” strand serves as a template that the determines the order of nucleotides along the new complementary strands.

DNA Replication The third step in replication
The nucleotides are connected to for the sugar/phosphate backbones of the new strands. Each DNA molecule now consists of one old strand and one new strand

DNA Replication Each DNA molecule completed in replication is identical to the parent molecule Term for the two daughter DNA molecules that are composed of one OLD strand and one NEW strand = SEMICONSERVATIVE

Semiconservative Model of Replication (diagram b)

How replication of DNA is carried out
A large team of Enzymes and other proteins carries out the process of DNA replication

Keep in mind… There are 46 DNA molecules (that is, chromosomes) in each of your cells That’s 6 billion base pairs It would take about 900 AP Biology books to print it all out (A’s, T’s, C’s and G’s) It takes a cell just a few hours to copy all of that information And the cells are VERY good at it – only 1 error per BILLION nucleotides, on average. AMAZING!

Directionality of DNA You need to number the carbons! nucleotide
it matters! nucleotide PO4 N base 5 CH2 This will be IMPORTANT!! O 4 1 ribose 3 2 OH

Replication in Prokaryotes and Eukaryotes
Replication of the DNA in bacteria and in the eukaryotic life forms is essentially similar, though there are some differences See discussion of origins of replication…

Replication – the Process
Origins of Replication Sites on the chromosome where replication begins A bacterial chromosome has only one origin of replication Eukaryotic chromosomes have multiple origin sites. This makes replication faster

What is an Origin of Replication?
An origin is a stretch of DNA that has a specific sequence of nucleotides Proteins that initiate DNA replication recognize this sequence and attach to the DNA at these points. This separates the DNA into two strands and opens up a replication bubble.

Origins of Replication - Diagram

What happens at a replication bubble?
Once a replication bubble is created, replication proceeds in both directions until the entire molecule is copied. In eukaryotic cells, the multiple (hundreds of thousands) of replication bubbles eventually fuse. This speeds up replication Replication proceeds in both directions from the origin.

What is a replication fork?
A replication fork is the Y-shaped region at either end of a replication bubble. New strands of DNA are being elongated at these points.

Antiparallel DNA Strands
Antiparallel means that sugar/phosphate backbones run in opposite directions.

Explanation of Antiparallel Strands
Remember the structure of the deoxyribose sugar in a nucleotide and the numbering of its carbons. Remember that the phosphate group is always attached to the 5’ carbon Remember that the phosphate group of the next nucleotide is always attached at the 3’ carbon

The result of nucleotides having distinct 5’ and 3’ ends is a DNA strand with distinct POLARITY When considering the long DNA strand at its 3’ end one would find a hydroxyl (-OH) group of the end nucleotide At its 5’ end would be the phosphate group of the end nucleotide

Sounds trivial, but… this will be IMPORTANT!!
5 The DNA backbone PO4 Putting the DNA backbone together refer to the 3 and 5 ends of the DNA the last trailing carbon base CH2 5 O 4 1 C 3 2 O –O P O Sounds trivial, but… this will be IMPORTANT!! O base CH2 5 O 4 1 3 2 OH 3

As it turns out, the two sugar/phosphate backbones that make up a DNA double helix are arranged “upside down” or antiparallel to each other.

Why Do We Care?? In a word…DNA POLYMERASE
What in the heck is DNA POLYMERASE?? DNA Polymerase is the enzyme that is in charge of building a new DNA molecule. The thing about DNA Polymerase is that, when building DNA, it can ONLY ADD NUCLEOTIDES TO THE PRE-EXISTING 3’ END OF AN ELONGATING DNA MOLECULE Think of the 3’ carbon as a “hook” Can’t hang a nucleotide onto a strand without a hook. Thus, A new DNA strand can elongate ONLY IN THE 3’ DIRECTION It can NEVER elongate in the 5’ DIRECTION WHY?? Has to do with the way the bonding of one nucleotide to another is energized. This has a big effect on what we see happen in the replication fork during REPLICATION.

So how does this DNA Replication happen?
A Large team of enzymes coordinates the steps of replication Here are the steps and the enzymes that cause them to happen Enzymes more than a dozen enzymes & other proteins participate in DNA replication Let’s meet the team…

Replication: 1st step Unwind DNA helicase enzyme
I’d love to be helicase & unzip your genes… Replication: 1st step Unwind DNA helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork

Replication: 2nd step Build daughter DNA strand
add new complementary bases DNA polymerase III does this! But DNA polymerase III has a PROBLEM DNA Polymerase III Oh, no…

DNA Polymerase III’s PROBLEM…
It can ONLY add to the 3’ end of an existing nucleotide. Well…if you just unzip a double helix of DNA, you will notice there are NO pre-existing nucleotides magically in place where the NEW replicate strands are going to be made. How the heck can you add to the EXISTING 3’ end of nucleotide if there’s no nucleotide there?? HEY! There’s NO NUCLEOTIDES IN HERE Not to seem uncaring, but…so what? Hmmm…that IS a problem…

Answer: Create a 3’ Hook…
5’ end Original Template Strand Answer: Create a 3’ Hook… Obvious question: Where the heck did the “hook” nucleotide come from? Who stuck THAT on there? Growing complementary strand Attaches to pre-existing 3’ hook nucleotide providing 3’ “hook” 3’ end

Step 2: Create a 3’ “hook” Original template DNA Actually Made of RNA,
3’ “Hook” is actually called a PRIMER. Actually a small bit of RNA (NOT DNA) Primer is laid down by an enzyme called PRIMASE Primase is the only enzyme that can add nucleotides to the bases of the ORIGINAL TEMPLATE strand of DNA Puts in place the 3’ “hook” needed for the creation and elongation of the NEW, complementary strands of DNA by DNA Polymerase III. Actually Made of RNA, NOT DNA The PRIMER “hook” Primase

but honestly…I really don’t
So WHY do we NEED a HOOK?? Shut up. It has to do with the way the bonding of one nucleotide to the next in the chain is ENERGIZED Um, not to seem unappreciative, but honestly…I really don’t think anyone cares…

Energy of Replication ATP TTP CTP GTP AMP ADP GMP TMP CMP
Where does energy for bonding usually come from? We come with our own energy! energy You remember ATP! Are there other ways to get energy out of it? energy Are there other energy nucleotides? You bet! And we leave behind a nucleotide! ATP TTP CTP GTP AMP ADP GMP TMP CMP modified nucleotide

Energy of Replication ATP GTP TTP CTP
The nucleotides arrive as nucleosides DNA bases with P–P–P P-P-P = energy for bonding DNA bases arrive with their own energy source for bonding They are bonded together by an enzyme: DNA polymerase III ATP GTP TTP CTP

The energy rules the process
PRIMER 5 3 Step 3: Elongation energy DNA Polymerase III Adding bases can only add nucleotides to 3 end of a growing DNA strand need a “starter” nucleotide to bond to strand only grows 53 energy DNA Polymerase III DNA Polymerase III energy DNA Polymerase III The energy rules the process. energy B.Y.O. ENERGY! The energy rules the process 3 5

How DNA Polymerase III works…
Because of the way the nucleotides energize their own bonding… Along ONE of the template (old/parent) strands of DNA: DNA Polymerase III can synthesize a CONTINUOUS complementary strand by elongating the new DNA in the 5’ to 3’ direction The strand made by this mechanism is called the LEADING strand.

How DNA Polymerase III works…
On the OTHER side of the replication fork, the process is DIFFERENT due to the way the nucleotides energize their own bonding. REMEMBER: DNA Polymerase III can ONLY add to the 3’ end of the elongating strand. On this 2nd template strand, because of the ANTIPARALLEL arrangement of the strands, DNA Polymerase III must work in the direction leading AWAY from the replication fork in order to “hang” a nucleotide onto a 3’ “hook”. The strand made in this direction is called the LAGGING strand. The lagging strand is made via DISCONTINUOUS synthesis

need “primer” bases to add on to
5 3 5 3 need “primer” bases to add on to energy no energy to bond  energy energy energy energy ligase energy energy 3 5 3 5

How the Lagging Strand is Made…
As the replication bubble opens, the DNA polymerase molecule can work away from the fork and make a short segment of DNA. As the bubble opens up a bit more, polymerase can leap frog back up the fork and slide back out of the fork again until it bumps into the strand it just made Thus, the lagging strand is made in a series of short segments Okazaki fragments nucleotides long in eukaryotes An enzyme must come back and “knit” these fragments together – it is called LIGASE.

More on Primers and Primase
Only 1 primer is required for the synthesis of the LEADING DNA strand. However, for the LAGGING strand, each new segment created must have its own primer And remember…these primers are made of RNA…can’t just leave RNA in the middle of the DNA…

Step 4: Replacing Primer with DNA
Another DNA Polymerase removes the RNA Primer and replaces it with DNA DNA Polymerase I Another enzyme makes the final “stitch” between the two DNA fragments that were once separated by the RNA primer Ligase

Here comes… A super duper recap of the processes of replication!

Leading & Lagging strands
Okazaki Leading & Lagging strands Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand  5 Okazaki fragments 5 5 3 5 3 5 3 ligase Lagging strand 3 growing replication fork 3 5 Leading strand  3 5 Lagging strand Okazaki fragments joined by ligase “spot welder” enzyme 3 DNA polymerase III Leading strand continuous synthesis

Replication fork / Replication bubble
5 3 3 5 DNA polymerase III leading strand 5 3 5 3 5 5 3 lagging strand 5 3 5 3 5 3 5 lagging strand leading strand growing replication fork growing replication fork 5 leading strand lagging strand 3 5 5 5

Starting DNA synthesis: RNA primers
Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand 5 5 3 5 3 5 3 3 growing replication fork 5 3 primase 5 DNA polymerase III RNA RNA primer built by primase serves as starter sequence for DNA polymerase III 3

Replacing RNA primers with DNA
DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I 5 3 ligase 3 5 growing replication fork 3 5 RNA 5 3 But DNA polymerase I still can only build onto 3 end of an existing DNA strand

Houston, we have a problem!
Chromosome erosion All DNA polymerases can only add to 3 end of an existing DNA strand DNA polymerase I 5 3 3 5 growing replication fork 3 DNA polymerase III 5 RNA 5 Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions? 3

Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions 5 3 3 5 growing replication fork 3 telomerase 5 5 Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG TTAAGGG 3

direction of replication
Replication fork DNA polymerase III lagging strand DNA polymerase I 3’ primase Okazaki fragments 5’ 5’ ligase SSB 3’ 5’ 3’ helicase DNA polymerase III 5’ leading strand 3’ direction of replication SSB = single-stranded binding proteins

DNA polymerase III enzyme
Roger Kornberg 2006 DNA polymerases DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal Arthur Kornberg 1959 DNA polymerase III enzyme In 1953, Kornberg was appointed head of the Department of Microbiology in the Washington University School of Medicine in St. Louis. It was here that he isolated DNA polymerase I and showed that life (DNA) can be made in a test tube. In 1959, Kornberg shared the Nobel Prize for Physiology or Medicine with Severo Ochoa — Kornberg for the enzymatic synthesis of DNA, Ochoa for the enzymatic synthesis of RNA.

Editing & proofreading DNA
1000 bases/second = lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases removes abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases

Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle

What does it really look like?
1 2 3 4

Replication and S phase of Interphase…
REMEMBER: This REPLICATION is what is happening at S in Interphase of the Cell Cycle!! Makes the SINGLE stranded chromosomes into DOUBLE stranded chromososomes. Chromosomes composed of TWO IDENTICAL chromatids

Any Questions??

DNA Replication 2007-2008.

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