Test 2: Chapters 5

DNA replication

parental strand DNA is the template for a new strand

• semi conservative process

• the primary enzyme is DNA polymerase

DNA polymerase

• main enzyme ofr DNA replication

• DNA polymerase catalyzes the doing of deoxyribonucleotide triphosphate 

• Joining of a new nucleotide on 3’ end of the primer strand that has a hydrogen bonded to the template

• and synthesis of the phos bond

• They are the most important enzyme because they are responsible for new nucleotides at the replication fork

What are the two fundamental properties of DNA polymerase?

1. Synthesize DNA only in the 5’ to 3’ direction

2. Add new dNTPs only to a primer strand that is hydrogen-bonded to the template

• both have to be satisfied in every DNA

DNA molecules grow in:

5’ to 3’ direction

DNA replication fork

• region of active DNA synthesis

• site of DNA replication, where there are two strand of parental DNA separating

• The two strand formed at the DNA replication fork serve as a template 

• Identified in E. coli

Leading Strand

this DNA strand is synthesized and read continuously in the 5’ to 3’ direction

Laggin Strand

this DNA strand is synthesized in short discontinuous pieces called Okazaki fragments

• in this strand the DNA polymerase backs up and synthesizes down to the end, then backs up and synthesizes down and continues doing it

Anti-parallel DNA

the 5’ end is on the same side as the 3’ end of another strand

• Once the two strands are split, the ends will be exposed leaving one with a 5’ end and one with a 3’ end

Okazaki fragments

short, discontinuous pieces of a DNA lagging strand that is being synthesized

DNA ligase

rejoins okazaki fragments

• this is done by catalyzing the synthesis of a phosphodiester bond

proteins at the replication fork

1. Sliding Clamp proteins

2. Clamp-loading proteins

3. Helicase

4. Single-Stranded DNA binding proteins 

Sliding Clamp Proteins

• loads DNA polymerase onto primer and helps maintains association with template 

• at the replication fork

Clamp-loading protein

• this protein loads sliding clamp protein onto replication fork

• at the replication fork

Helicase

• catalyzes unwinding of parental DNA

• breaks hydrogen bonds that are connecting the double stranded DNA

• at the replication fork

Single-Stranded DNA binding protein

• stabilizes unwound template DNA so that it is available to the polymerases

• binds to a single exposed strand of DNA

• helps keep the single strand of DNA stable so the single strand can be accessible to the DNA polymerase

• this happens once the helicase separates the strands

• at the replication fork

Topoisomerase

• catalyze transient breaks in DNA strands

• Acts as a swivel point

• This protein travels ahead of the replication fork 

• It helps relieve treason that is introduced in the DNA molecule by clipping the phosphodiester backbone, lets the DNA spin to relieve tension, and then repair the break 

Proofreading activity of DNA polymerase

• requires DNA primers

• Catalyze growth in 5’ to 3’ direction

• 3’ to 5’ exonuclease activity to remove a base pair if there is a mistake (so it can go backwards if there is a mistake)

Origin of Replication

Ori

Maintaining the Ends of Chromosomes

Telomeres

• these are terminal sequences of linear DNA molecules

• this means it is only in eukaryotes

• it has to be maintained for the chromosome integrity because telomeres help prevent degradation

• telomeres cannot be replicated 

Telomerase

• synthesize or replicate telomeres to help maintain chromosome length

• catalyze synthesis of telomeres without DNA template

• is a reverse transcriptase that carries its own RNA template

• telomerase extends 3’ end

What sequence does telomerase add?

telomeric repeats: TTAGGG to the ends of DNA

Polymerase a (alpha)

• primase complex synthesis the complementary strand

• this helps keeps the end long enough to protect the rest of the sequence of the chromosome

What helps maintain telomere length?

• telomerase activity

premature aging syndrome of telomeres:

• rate of telomere loss is to high

Telomeres with Cancer cells

• high levels of telomerase because it allows for continued cell division

What are two causes of DNA mutation?

1. Incorporation of incorrect bases during DNA replication
2. Occur spontaneously or from exposure to chemicals/radiation

What are the effects on DNA of damage/mutations?

1. Block replication or transcriptions
2. Leads to high frequency of mutations

What are two types of DNA repair?

1. Direct Reversal of the chemical reaction responsible for DNA damage (selective of what cells use this)

2. Excision Repair: (widely used)
   - removal of damaged bases
   - replacement of newly synthesized DNA

Pyrimidine Dimer

• UV damage forms this

• it distorts the structure of DNA and block transcription or replication

Photoreactivation

• repairs pyrmidine dimer damge

• energy from visible light breaks the cyclobutane ring structure leading to reversing the dimerization reaction

What are the four types of excision repair:

1. Base-excision repair

2. Nucleotide-excision repair

3. Transcription-coupe repair

4. Mismatch repair

Base excision repair

• base excision repair is when a single damaged base are recognized and removed

What cleave the bind between uracil and deoxyribose of the DNA

DNA glycosylase

What clips the back bone to remove the sugar and phosphate (the phosphodiester bond) and breaks DNA/RNA into smaller fragements?

endonucleases

What is the purpose of ligase?

repairing and connecting DNA or RNA strands by forming covalent phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate ends

DNA lesions blocks _________ becuase it cannot be copied like normal DNA polymerase

DNA replication

A pyrimidine dimer is a _____

translesion

What are the required enzymes for nucleotide-excision repair in bacteria?

1. helicase - to access region of mutation

2. exconuclease - clips DNA backbond to cut out oligonucleotide

3. DNA polymerase and ligase - replace and fix what is removed

What are the two repair mechanisms of double-stranded breaks in DNA?

1. Non-homologous end joint

2. Homologous recombination

Two reasons for DNA recombination:

1. control gene expression

2. contribute to genetic diversity

What is the structure of immunoglobulins like?

• there is a pair of identical heavy and light polypeptide chains

• C-terminal = constant regiona

• N-terminal = variable region

Rearrangmeent of Light chain Gene on Immunoglobulin

1. V region: n-terminal amino acids of variable region

2. J region: C-terminal amino acids of the varaible regional

3. C region: encodes the constant region

Heavy Chain gene on immunoglobulin rearrangement:

1. D region encodes amino acids between V and J regions

2. Two recombination events:
   1. D recombins with J
   2.