MBIO 2710 / Topic 11: Nucleic Acid & Pr

Describe each of the three models for DNA replication.

Conservative model

> One completely old w/ two parent strands + one completely new w/ two daughter strands (both identical). Implies re-association of parental strands post-replication.

Semi-conservative model

> Each DNA has one continuous parental strand and one continuous daughter strand.

Dispersive model

> Each DNA has pieces of parental and daughter strand. Implies cleavage of parental strands during replication and subsequent reassembly.

Explain the 1958 Meselson and Stahl experiment.

> Used N because of the abundance of N in bases.

> Grew E. coli in media containing ¹⁵NH₄Cl (ammonium chloride) as soul source of N.

> Cells generate NAs and nts with heavy nitrogen and not light nitrogen.

> Added these E. coli cells to media containing ¹⁴NH₄Cl.

> Sampled cells at various time-points with DNA extraction.

> The particles that has density similar to a specific region in the tube will be positioned around that specific region.

> ¹⁴N DNA, whose density is light, will be around the region with light density.

> ¹⁵N DNA, whose density is heavy, will be around the region with heavy density, allowing DNA replication model analysis.

After one full replication, which replication model was eliminated, and why?

> Photographed result showed one half-heavy, half-light band.

> Indicates that both synthesized DNA molecules had half-light and half-heavy nitrogen.

> Disproves the conservative model, which states that one synthesized DNA molecule is full light and one synthesized DNA molecule is full heavy.

After two full replications, which replication model was eliminated, and why?

> Photographed result showed one band w/ light DNA and one band w/ half-heavy DNA.

> Indicated that two of the total DNAs had half-light and half-heavy.

> Indicated that other two of the total DNAs had full light.

> Disproves the dispersive model, which states that all four total DNA molecules would have ¼ ¹⁵N and ¾ ¹⁴N.

How many DNAPs does one cell usually have?

Each cell has > 1 DNAP.

What are the 5 commonalities for DNAPs?

> Uses dNTPs.

> Requires a primer before elongation.

> Primer elongation directed by template.

> Incorporating two strands together require a template.

> Makes 5’ to 3’.

What does “recognizes 2’H and adds to 5’ end” mean in DNA synthesis?

> 2’H = hydrogen on the 2’ carbon of sugar, distinguishing it from RNA’s 2’OH.

> Enzymes recognize 2’H to identify DNA nts.

> During DNA synthesis, nts added by linking 5’ phosphate of incoming nucleotide to the 3’ OH of the growing strand.

What does “need to break to monophosphate to combine in DNA” mean?

> nts for DNA synth usually come as dNTPs.

> When added to DNA, two terminal phosphates (pyrophosphate) are cleaved off.

> This leaves the monophosphate (attached to sugar) that actually links into the DNA strand.

Note: Breaking off pyrophosphate drives the reaction forward (irreversible).

Why does a primer prevent DNA loss during replication?

> DNA polymerase requires an existing 3’ OH to add nucleotides; it cannot start DNA synthesis de novo.

> RNA primer provides the initial 3’ OH, allowing replication to begin.

> Without primers, the ends of linear DNA (telomeres) would shorten each replication cycle.

> Primers prevent loss of genetic information by ensuring full replication of DNA ends.

Differentiate the phrases template strand, non-template strand, non-coding strand, and coding strand in a way that makes sense in respect to RNA pol activity.

> RNAP reads only one strand of DNA (the template strand). The other strand is not read (the non-template strand).

> RNAP reads one strand that does not have the code for the protein to be made (the non-coding strand). The other strand has the code for the protein (the coding strand).

Why is the template/non-coding strand read instead of the non-template/coding strand?

> What we want is to copy the gene.

> If we were to copy the coding strand, then we would get a copy of base sequences that the gene would base-pair to and not a copy containing the bases of the gene.

> We need to get a copy containing the bases of the gene.

> The only way to do that is to copy the non-coding strand.

> If we read the non-coding strand, then we would be able to get the bases of the coding strand, which is what we want.

What are the 8 main steps in DNA replication?

> RNAP (primase) lays down RNA primers on both strands

> DNAP extends DNA from RNA primers.

> Leading strand synthesized continuously.

> Lagging strand synthesized discontinuously as Okazaki fragments.

> Multiple replication origins initiate replication simultaneously.

> RNase removes RNA primers.

> DNAP fills gaps left by primers.

> DNA ligase seals nicks by creating phosphodiester bonds.

Why does DNA replication occur bidirectionally, with forks working on both sides to meet in the middle?

> DNA replication starts at multiple origins and proceeds in both directions from each origin.

> Working on both strands simultaneously shortens replication time.

> Replication forks move away from the origin and eventually meet in the middle, completing DNA duplication efficiently.

Why does DNA replication occur bidirectionally, with forks working on both sides to meet in the middle?

> DNA replication starts at multiple origins and proceeds in both directions from each origin.

Working on both strands simultaneously shortens replication time.

Replication forks move away from the origin and eventually meet in the middle, completing DNA duplication efficiently.