1. Cell Biology Review
Cinderella Aquino
3-2-10

2. Cell Cycle • M (mitotic) phase
The cell cycle consists of highly ordered events that result in the duplication and division of a cell
• M (mitotic) phase
- chromosome segregation, cytoplasmic division
• Interphase
• G1 (Gap 1)
- transition from M to S phase
- synthesis of proteins that would be required during S phase
- G0: Cells that become arrested in G1 (quiescence)
• S (synthetic) phase
- DNA synthesis
• G2 (Gap 2)
- transition from S to M
- synthesis of proteins needed during M phase
S phase is where you duplicate your DNA.
G1 is the longest phase, mitosis is the shortest phase.
When are the histones made?
Will be learning the regulation of the cell cycle later and this is important in knowing how cancers develop. There are checkpoints between the phases to regulate growth and if these checkpoints are not regulated, then cancer will likely develop.

3. Three Key Components of Nucleotides
Nitrogenous Base
Phosphate
Ribose Sugar
Know what attaches to each C.
What is the difference between ribose and deoxyribose and dideoxyribose sugars? Which of these are utilized in zidovudine (ZDV)?
Where do you add another nucleotide and what kind of bond does it form?
What bases pair and what kind of bonds and how many do they form? (Think back to melting temperatures)

4. Nitrogenous Bases Purines Pyrimidines Double ring structure
Adenine, Guanine
Pyrimidines
Single Ring Structure
Cytosine & Thymine (DNA only)
Uracil (RNA only)
Uracil is not in DNA because cytosine can spontaneously convert into uracil
Repair enzymes recognize uracil in DNA, excise, replaced with cytosine.
What is the result when cytosine is methylated?
What is the result when cytosine is deaminated?
What is the result when cytosine is methylated, then deaminated?
This is important to know when talking about DNA repair mechanisms and how it leads to disease.
Very important to know, likely to be on the exam.

5. DNA Replication
DNA undergoes semiconservative replication (half of the parent molecule is retained by each daughter molecule)
Semiconservative replication of DNA: The two strands of parental DNA separate, and each serves as a template for synthesis of a new daughter strand by complementary base pairing.
Figure 5-2 Molecular Biology of the Cell (© Garland Science 2008)
In what direction is DNA synthesized?
How are leading and lagging strands synthesized differently?
Check out the silent video at

6. DNA polymerases catalyze the elongation of DNA chains
In the DNA polymerase reaction, incoming nucleotides are covalently bonded to the 3’ hydroxyl end of the growing DNA chain.
Each successive nucleotide is linked to the growing chain by a phosphoester bond between the phosphate group on its 5’ carbon and the hydroxyl group on the 3’ carbon of the nucleotide added in the previous step (5’-> 3’)
Figure 5-3 Molecular Biology of the Cell (© Garland Science 2008)
Key Concept:
DNA synthesis directional
(5’ to 3’ only)!!!!

7. Proofreading is performed by the 3’ -> 5’ exonuclease activity of DNA polymerase
Incorrect nucleotide incorporated about 1 in 100,000 nucleotides. Cell needs process to lower mutation rate
Proofreading mechanism
Exonuclease activity chews away misincorporated nucleotide
Figure 5-9 Molecular Biology of the Cell (© Garland Science 2008)
What would happen if this proofreading activity was defective?

8. Eukaryotic and Prokaryotic DNA Polymerases
The main types of bacterial DNA Polymerases (I, II, III, IV & V)
DNA Polymerase III is the primary polymerase
Polymerase I is responsible for removing RNA primers, proofreading, DNA repair and filling in gaps left by DNA pol III
The main types of eukaryotic DNA polymerases (a,d, g & e)
Polymerase g found only in mitochondria
Polymerase a is involved with initial synthesis of DNA strands off RNA primers
Polymerase d & e synthesizes leading & lagging strands
The polymerases that have Roman numerals are prokaryotic and the ones with Greek letters are eukaryotic.
This rule only applies to DNA polymerases! Will see differently in RNA polymerases when talking about transcription.
Know all of these, will likely see an exam question on this. If not on this exam, then in the future.

9. DNA replication is usually bidirectional
Origin of Replication
Starting site for DNA synthesis
Prokaryotic genomes usually have a single origin of replication
Most circular DNA molecule are replicated in a bidirectional process from a single origin.
This Theta replication occurs in bacteria, mitochondria, chloroplasts and some viruses
Prokaryotic replication origin termed ori
Replication Forks
Structure formed when DNA strands are separated at site of DNA replication
Bidirectional replication of a circular chromosome. Replication begins at the point of origin (oriC) and proceeds in both directions at the same time.
Compare this to eukaryotic replication

10. Eukaryotic DNA replication involves multiple replicons
Replicons: units of replication on linear DNA
Typical chromosome may contain several thousand replicons
In Yeast:
Autosomously replicating sequence (ARS element)
Large number of replicons allows eukaryotics to replicate their DNA faster
Remember the differences between prokaryotic and eukaryotic replication. Make a table if you need to.

11. Eukaryotic Pre-replication Complex
Initiation process requires formation of pre-replication complex
Origin Recognition Complex (ORC) binds to replication origin
The MCM complex including helicase binds next
Requires assistance from helicase loaders
The DNA has been “licensed” for replication
The completed complex: Pre-replication Complex
The process of licensing makes sure that the cells only replicated their DNA only once
Figure Molecular Biology of the Cell (© Garland Science 2008)

12. First Set of Steps in DNA Replication
Figure Molecular Biology of the Cell (© Garland Science 2008)
Single stranded binding proteins: bind to keep DNA from reannealing
Topoisomerase: controls supercoiling
DNA primase to lay RNA primer: DNA can’t lay down from scratch

13. Unwinding DNA double helix requires helicases, topisomerases, and single-strand binding proteins
Helicase: unwinds DNA
Topoisomerase: decreases supercoiling caused by unwinding
Gyrase: a type II topoisomerase used by bacteria
Single-strand binding proteins: bind to exposed single strands and stabilizes the DNA for DNA replication

14. RNA primers initiate DNA replication
DNA polymerase MUST have a 3’-OH on the target in order to add a nucleotide
DNA polymerase cannot start synthesis from scratch
However, RNA polymerases can
Primase: an enzyme that synthesizes RNA fragments about 10 base pairs long using DNA as a template
In bacteria, primase part of a complex called primosome
In eukaryotic cells, primase is tightly bound to DNA polymerase a (initiates DNA replication)
RNA segment is removed when the neighboring growing strand reaches the primer stretch
DNA primase is an RNA polymerase needed to start DNA replication since it can start from scratch.
What happens to RNA primers after it’s completed it’s function? More specifically, how does it get removed?

15. Prokaryotic vs Eukaryotic Priming
Again, notice the difference here. Know which polymerases are used in which process.
DNA polymerase / 

16. Leading and Lagging strands
DNA polymerase can only synthesize DNA in the 5’ to 3’ direction
Two types of newly synthesized strands
The leading strand is synthesized continuously in the direction of replication fork movement.
The lagging strand is synthesized in small pieces (Okazaki fragments) backward from the overall direction of replication.
The Okazaki fragments are then joined by the action of DNA ligase.
Figure 5-7 Molecular Biology of the Cell (© Garland Science 2008)
The lagging strand is synthesized in small pieces because it needs to wait for the replication fork to open up.

17. Ligase activity: Joining of nick strands together
Figure Removal of RNA primer and filling of the resulting “gaps” by DNA polymerase I.
Fig Action of DNA ligase. Two polynucleotide chains, one with a free 3′-OH group and one with a free 5′-phosphate group, are joined by DNA ligase, which forms a phosphodiester bond.
Figure Molecular Biology of the Cell (© Garland Science 2008)
Figure Molecular Biology of the Cell (© Garland Science 2008)
Figure Molecular Biology of the Cell (© Garland Science 2008)

18. A sliding clamp holds moving DNA polymerase onto the DNA
The sliding clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-stranded region of DNA
Sliding clamp forms a ring-like structure around DNA
The assembly of the clamp around the DNA requires a clamp loader
The clamp loader hydrolyzes ATP as it loads the clamp on to a primer-template junction.
Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008)
If you see a lot of PCNA, it could be a cancer or tumor bc of hyperproliferation of replication.
Proliferating Cell Nuclear Antigen, commonly known as PCNA, is a protein that acts as a processivity factor for DNA polymerase δ in eukaryotic cells. It achieves this processivity by encircling the DNA, thus creating a topological link to the genome. It is an example of a DNA clamp.
Prokaryotes: beta-subunit clamp
Eukaryotes: PCNA

19. Summary of DNA Replication
-MUST KNOW -
RPAs are SSBs in eukaryotes
RNA primers are destroyed by DNA polymerase I. It has 3’ to 5’ exonuclease activity.
Remember: what is a primase? What is a gyrase? What is the role of the single stranded binding proteins?
How are the leading and lagging strands synthesized differently? What is the meaning of semiconservative replication here?

20. Eukaryotic Replication Fork
DNA polymerase in eukaryotes does not associate into a dimeric complex
2 copies of polymerase remain separate
Replication protein A (RPA) binds the ssDNA preventing reannealing
Replication factor C (RFC) induces binding of proliferating cell nuclear antigen (PCNA)
Chromatin remodeling proteins are also involved with eukaryotic DNA replication
They help move nucleosomes
Compare Eukaryotic and Prokaryotic complexes.
Flap endonuclease 1 is an enzyme that in humans is encoded by the FEN1 gene.[1][2]
The protein encoded by this gene removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. D

21. Telomerase
Action of telomerase Telomeric DNA is a simple repeat sequence with an overhanging 3 end on the newly synthesized leading strand. Telomerase carries its own RNA molecule, which is complementary to telomeric DNA, as part of the enzyme complex. The overhanging end of telomeric DNA binds to the telomerase RNA, which then serves as a template for extension of the leading strand by one repeat unit. The lagging strand of telomeric DNA can then be elongated by conventional RNA priming and DNA polymerase activity.
DNA polymerase fills in the gap bc telomerase extends the overhang. Create a 3D protection by looping
What is telomerase role in cancers?

22. Some Key Differences Between Eukaryotic & Prokaryotic DNA Synthesis
Eukayotes have multiple origins versus single origin in prokaryotes
Eukaryotes undergo DNA synthesis during S phase in cell cycle
Eukaryotes remove RNA primers using FEN1 and RNaseH
Eukaryotes have telomeres at the ends of their linear DNA molecules, prokaryotic genomes are usually circular and do not have telomeres
Make tables to help.

23. Types of DNA Damage DNA replication Mismatches mismatches Base
alteration
2. Chemicals (nitrous acid) UV
Thymine
dimer
3. Radiation
Come back to this slide after reviewing the information in the following slides. Go through each type of damage of recall which diseases exemplify each mechanism and what enzymes are involved.
Double strand
break
high-energy
radiation
4. Spontaneously
loss of nucleosides
Base
alteration

24. Figure 5-47 Molecular Biology of the Cell (© Garland Science 2008)
AP endonuclease repair to fix depurination
Uracil glycosylase fixes deamination
Consequences if deamination (A), or depurination (B), damaged DNA were NOT repaired

25. Deamination of DNA Nucleotides
Cytosine deaminates to uracil
Methylated cytosine deaminates to thymine
Role of methylated cytosine in gene expression? Heterochromatin or euchromatin? What mechanisms repair these? What enzymes are involved?
Figure 5-50a Molecular Biology of the Cell (© Garland Science 2008)
Figure 5-50b Molecular Biology of the Cell (© Garland Science 2008)

26. Inherited Defects in DNA Repair Cause Known Diseases
Maintaining the integrity of the genome is extremely important for our survival
Mutations in genes involved with DNA repair can increase the risk of cancer and developmental abnormalities
MUST KNOW!!!! HNPCC results from a mutation in DNA mismatch repair
Hereditary nonpolyposis colorectal cancer (HNPCC) is a colon cancer, characterised by a risk of other cancers of the endometrium, ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain, and skin. The increased risk for these cancers is due to inherited mutations that impair DNA mismatch repair.
Table 5-2 Molecular Biology of the Cell (© Garland Science 2008)

27. DNA Repair Mechanisms
Translesional synthesis: DNA synthesis of new DNA across regions in which the DNA template is damaged
DNA polymerase eta can catalyze DNA synthesis across regions with thymine dimers
Excision repair pathways
Base excision repair: repair single damaged bases
Nucleotide excision repair: repair major distortions in DNA double helix
Pathway that corrects thymine dimers
Mutations in pathway causes Xeroderma pigmentosum

28. Uracil-Glycosylase Removes Uracil From DNA
When DNA is damaged through deamination reactions, cytosine is converted to uracil
Error repaired by uracil-DNA glycosylase
Damage repair helps explain why DNA contains thymine instead of uracil
If uracils were normally found in DNA then DNA repair would not be able to distinguish normal uracils from deamination generated uracils

29. AP Endonucleases Two Key Roles of AP Endonuclease:
Repair nucleotides missing bases caused by spontaneous events, such as depurination
Repair nucleotides missing bases caused by target repair process, such as by uracil glycosylase

30. Nucleotide Excision Repair (NER)
NER corrects problems such as pyrimidine dimers
Steps in NER
1. Identification of the mismatched or mutated DNA strand
2. Nick the mismatched DNA or mutated strand by endonuclease
3. 5’-3’ DNA polymerase fill the gap,
4. DNA ligase forms phosphodiester linkage
Important to remember when studying Xeroderma Pigmentosa

31. NER Diseases
Human genetic disorders associated with defects on nucleotide excision repair:
Xeroderma Pigmentosum (XP), Cockayne syndrome, Trichothiodystrophy
XP characterized by sun sensitivity, ocular involvement, > 1000-x increased risk of cutaneous and ocular neoplasms. ~50% of affected individuals demonstrate acute sun sensitivity from early infancy, acquiring severe sunburn with blistering or persistent erythema on minimal sun exposure; marked freckling of sun-exposed areas in a child before age two years is typical of XP
Very important, will learn in genetics too

32. Mismatch Repair Prokaryotes Eukaryotes
Normal, undamaged, but mismatched bases bind proteins of the mismatch repair system.
In bacteria, these proteins recognize the older, parental strand because it is methylated and replace a segment of newly synthesized (and unmethylated) DNA containing the mismatched base.
The mechanism for distinguishing between parental and newly synthesized strands in humans is not as well understood.
Scan for nicks in the new strand
Eukaryotes
Figure 5-20a Molecular Biology of the Cell (© Garland Science 2008)

33. Human Mismatch Repair and HNPCC
Hereditary nonpolyposis colon cancer (HNPCC) results from mutations in genes coding for proteins involved in mismatch repair
Most common mutations:
MLH1 and MSH2
HNPCC: Hereditary Non-Polyposis Colon Cancer
IMPORTANT TO REMEMBER: Will see this in genetics too and you will learn that MLH1 and MSH2 are part of the caretaker group of proteins.
~40%
~50%
~10%
Adapted From Peltomaki (2001) Human Molecular Genetics 10:735

34. Mutation ‘hot spots’
The occurrence of point mutations in the genome is not necessarily a random event.
30% of point mutations in known inherited diseases, involve C → T transitions (G → A, in the opposite strand), at sites containing CG dinucleotides (the so called CpG islands)
The CG doublet represents a true ‘hotspot’ for mutation in human genome.
Transitions occur more frequently at CpG islands, because the cytosine is prone to methylation at position 5, & spontaneous deamination of 5-methylcytosine to thymine follows [ C→ 5-MethylC→T ]
C→T transition in one strand is matched in the opposite strand by G →A transition
Again, what happens when cytosine is methylated and what happens to gene expression? This is an important basis for learning about diseases in genetics.

35. Repair of Double-Strand DNA Breaks
Double-strand DNA Breaks are repaired by nonhomologous end-joining or homologous recombination
Nonhomologous end-joining uses a set of proteins that bind to the ends of the two broken DNA fragments and joins them together
Error-prone
A typical somatic cell in a 70-year old had over 2,000 end-joining repairs
Homologous recombination
The intact chromosome acts as a template to guide repair of the damaged chromosome
Breast Cancer genes BRCA1 & 2 involved with this pathway
End joining : glue back together. Error prone process
Homologous recombination: more accurate

36. Homologous Recombination
Nonhomologous
End-Joining
Homologous Recombination Repairs Spontaneous Breaks in DNA and Those Induced in Meiotic Crossing-over
Ku heterodimers grasps the broken chromosome ends and help recruit other proteins to fix the break
Figure 5-52a Molecular Biology of the Cell (© Garland Science 2008)

37. Hereditary Breast and Ovarian Cancer Breast Cancer Susceptibility Genes BRCAs
BRCA1 and BRCA2 are tumor suppressor proteins
Abnormal BRCA1: dominant susceptibility gene- confers high risk of breast and ovarian cancer (Type 1).
BRCA1 breast cancer affects 1:800 women in the U.S.
Jewish women of eastern European descent, the risk is 1:100
BRCA2 gene confers a high risk of breast cancer, not an elevated risk of ovarian cancer. (Type 2).
Mutations also increase risk of prostate, pancreatic, gall bladder, and male breast cancers.
Mutations in these genes predispose a woman to breast cancer. BRCA2 gene mutations are found twice as much than BRCA2 mutations. Also IMPORTANT TO REMEMBER for Genetics.
So, if these genes are tumor suppressors, would the upregulation or downregulation of these genes be involved in causing cancer?

38. Figure Key concept map for DNA structure, replication, and repair. NHEJ = nonhomologous end-joining; HR = homologous recombination.
Look at this concept map and see if you can draw it out yourself. Everything here is very important for success in genetics!!!