Ch. 14 DNA Structure & Function

Which nitrogenous bases are purines?

Which nitrogenous bases are purines?

Adenine & guanine

Which nitrogenous bases are pyrmidines?

Cytosine, thymine, and uracil

What is the name for the DNA backbone?

Phosphodiester backbone

DNA-interacting proteins can fit in ___________ to “read” specific DNA sequences

Major & minor grooves

Guanine forms how many hydrogen bonds with cytosine?

3

Adenine forms how many hydrogen bonds with thymine?

2

What gives consistent diameter of helix?

Equal bond distance

What were the 3 possible models of DNA replication?

Conservative, semiconservative, dispersive

Which of the 3 models of DNA replication is supported by the Meselson & Stahl experiment?

Semiconservative

What are the 3 things required for DNA replication?

Parental DNA strand (template), enzymes, and nucleotide triphosphates

What are the 3 steps of DNA replication?

1. Initiation

2. Elongation

3. Termination

What matches existing DNA bases with complementary nucleotides and links them?

DNA polymerase

What are the features of DNA polymerase?

1. Add new bases to 3’ end of existing strands

2. Synthesize in 5’-3’ direction

3. Requires a primer (usually RNA)

Which polymerase makes primer?

RNA polymerase

Which polymerase extends primer?

DNA polymerase

Single circular molecule of DNA. Begins at origin of replication. Proceeds in both directions around the chromosome

Prokaryotic Replication

What enzyme in prokaryotic replication clamps onto and unwinds DNA?

Helicase

Unwinding DNA in one area will increase tension in the rest of the strand, resulting in what?

Supercoiling (one complete helical turn = one supercoil)

Which enzyme unwinds DNA to prevent supercoiling?

Topoisomerases

Which specific topoisomerase is used in replication?

DNA gyrase

Partial opening of the helix forms ________________ _____________

Replication forks

What is a type of RNA polymerase that makes RNA primer?

DNA polymerase

Label

1. Continuous. Leading strand

2. Discontinuous. Lagging strand

3. Discontinuous. Lagging strand

4. Continuous. Leading strand

The main replication enzyme out of the 3 in E. coli

DNA polymerase III

The enzyme that has the 5’-3’ exonuclase activity. Acts on lagging strand to remove primers and replace them with DNA

DNA pol I

The enzyme involved in DNA repair processes

DNA pol II

What is synthesized continuously from a single initial primer and extended by DNA pol III?

Leading strand

Subunit of DNA pol III forms “sliding clamp” to keep it attached to the DNA template

Processivity

What loads clamp onto DNA?

Clamp Loader

What is synthesized discontinuously with multiple priming events?

Lagging strand

In the lagging-strand synthesis process, what produces an RNA primer for each Okazaki fragment?

DNA primase

In the lagging-strand synthesis process, what synthesizes DNA from each RNA primer?

DNA pol III

In the lagging-strand synthesis process, what seals the DNA backbone?

DNA ligase 1

Enzymes at each replication fork (in prokaryotic replication) form a large macromolecular assembly called what?

Replisome

What are the 2 main components of the replisome?

1. Primosome

2. Complex of 2 DNA pol III (one for each strand)

What does the primosome consist of?

All the proteins involved in DNA synthesis (primase, helicase, clamp loader)

A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand

1st step in prokaryotic replication

The “loop” in the lagging-strand template allows replication to occur 5’-3’ on both strands, with the complex moving to the left

2nd step in prokaryotic replication

When the DNA pol III on lagging strand hits the previously synthesized fragment, it releases the B-clamp and template strand. DNA pol I attaches to remove the primer

3rd step in prokaryotic replication

The clamp loader attaches the B-clamp and transfers this to DNA pol III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA pol I removes the primers

4th step in prokaryotic replication

After B-clamp is loaded, DNA pol III on lagging strand adds bases to the next Okazaki fragment

5th step in prokaryotic replication

What are short sections of DNA formed at the time of discontinuous synthesis of the lagging strand during replication of DNA?

Okazaki fragments

Occurs at a specific site roughly opposite of origin of replication. 2 intertwined daughter molecules are produced (chromosomes are eventually unliked by DNA gyrase)

Termination

How is eukaryotic replication more complicated than prokaryotic?

1. Larger amount of DNA in multiple chromosomes to copy

2. Linear structure. Need to deal with ends

How many origins must eukaryotic replication require?

Multiple

True or false: eukaryotic origins of replication are sequence-specific, they cannot be adjusted

False (they can be adjusted. Not sequence specific)

Each (eukaryotic & prokaryotic) origin on replication opens up how many times per DNA replication cycle?

Once

How is the initiation process in eukaryotic replication more complex than prokaryotic?

1. Requires more factors

2. Primase is a little more complex

How does eukaryotic replication initiation require more factors?

1. Need to assemble helicase and primase complexes onto template

2. Need to load polymerase with sliding clamp unit

How is primase more complex in eukaryotic replication initiation?

1. Complex of an RNA polymerase and a DNA polymerase

2. First makes short RNA primers then extends with DNA

How is the elongation process in eukaryotic replication more complex than prokaryotic?

Main replication polymerase is a complex of 2 DNA polymerases

Just know these ☹

Got it 😃

Most of these are alluded throughout the KnowIt, but just know these ☹

Got it 😃

Why do we have to worry about the ends on eukaryotic DNA?

We have nucleases in our cells that recognize and chop up free DNA. The ends protect against viruses so we don’t want them chopped up!

What are specialized structures found on the ends of eukaryotic chromosomes that protect them from nucleases and maintain the integrity of linear chromosomes?

Telomeres

What are telomeres composed of?

Short repeated sequences of DNA

What is the enzyme that makes telomere by using an internal template (not the DNA itself) and makes repeated copies of telomere by sliding?

Telomerase

What happens/forms at the ends of telomeres?

Forms a short overhang with same sequence which eventually forms a loop

What happens when telomeres are gone?

1. Important genes may be lost

2. Cell might think the end is a DNA double-strand break and try to repair it

3. Aging/cell death

What type of cells show upregulated telomerase activity?

Cancer cells

What is any agent that increases the number of mutations above background level?

Mutagen

What are the 2 categories of DNA repair systems?

1. Specific DNA repair

2. Nonspecific DNA repair

Which type of repair system targets a single kind of lesion in DNA and repairs only that damage?

Specific DNA repair

Which type of repair system uses a single mechanism to repair multiple kinds of lesions in DNA?

Nonspecific DNA repair

A specific repair mechanism that only repairs damage caused by UV light - creation of thymine dimers

Photorepair

What are the covalent linkage of adjacent thymine bases in DNA called?

Thymine dimers

What is an enzyme that uses energy from visible light to cleave thymine dimers?

Photolyase

A nonspecific repair mechanism where the damaged region is removed and replaced through DNA synthesis

Excision repair

What are the 3 steps of excision repair?

1. Recognition of damage

2. Removal of the damaged region

3. Resynthesis using the information on the undamaged strand as a template

Which type of repair process is a last-ditch effort when you just stick in any base and hope cell survives? (In E. Coli, called SOS response)

Error-prone repair

Which type of repair process is also used during crossing over in meiosis?

Double-stranded break repair

What biotechnology makes multiple copies of a piece of DNA and can be used to clone DNA, amplify small amounts of DNA to detectable levels, sequence DNA, and diagnose genetic diseases?

PCR (Polymerase Chain Reaction)

Denaturation. 95 degrees. Incubate target DNA at high temp to separate the strands. Add primers, nucleotides, and DNA polymerase

1st step in PCR

Annealing. 50 degrees. Primers attach to single-stranded DNA during incubation at low temp

2nd step in PCR

What is the 3rd step in PCR?

Extension. 72 degrees. Incubate at intermediate temp; DNA polymerase copies the target DNA

What are enzymes that cleave DNA at specific sites and are used by bacteria to destroy viral DNA?

Restriction endonucleases

The cut sites where restriction endonucleases cut?

Palindromes

What is the result of a cut in the middle of a palindrome, resulting in no overhang?

Blunt ends

What is the result of a staggered cut resulting in an overhang?

Sticky ends

True or false: Any 2 DNA molecules cut with the same restriction endonuclease can be joined together

True

Restriction endonucleases allow for the creation of ____________ ______ (from two different sources)

Recombinant DNA

What enzyme joins two fragments forming a stable DNA molecule, catalyzes formation of a phosphodiester bond, and join Okazaki fragments in replication?

DNA ligase 2

What is the introduction of DNA from an outside source into a cell? Temperature shifts can induce this in E.Coli

Transformation

What is an organism that is partly or entirely made from transformed cells?

Transgenic organisms

What is a genetically indentical copy called?

Clone

What is the isolation of a specific DNA sequence (usually protein-encoding) called?

Molecular (gene) cloning

What is the most flexible and common host for cloning?

E. Coli

What carries DNA in host and can replicate in the host?

Vector

What are small, circular chromosomes used for cloning small pieces of DNA?

Plasmids

What are the 3 components of plasmids?

1. Origin of replication

2. Selectable marker

3. Multiple cloning site (MCS)

What is the origin of replication’s role in plasmids?

Allows for independent replication

What is the selectable marker’s role in plasmids?

Allows the presence of plasmid to be easily identified

What is the multiple cloning site’s role in plasmids?

Unique restriction enzyme sites to easily clone in genes