DNA Replication

Nucleosome

A nucleosome is a section of DNA that is wrapped around a core of proteins

Proofreading

Ability of DNA polymerases to remove and replace incorrectly paired nucleotides in the course of replication. When an incorrect nucleotide is added to the growing strand, replication is stalled by the fact that the nucleotide's exposed 3′-OH group is in the "wrong" position During proofreading, DNA polymerase enzymes recognize this and replace the incorrectly inserted nucleotide so that replication can continue

Bacterial DNA Replication: Initiation and Unwinding Proteins

DNA helicases are responsible for breaking the hydrogen bonds that join the complementary nucleotide bases to each other Because the newly unwound single strands have a tendency to rejoin, another group of proteins, the single-strand-binding proteins, keep the single strands stable until elongation begins. A third family of proteins, the topoisomerases, reduce some of the torsional strain caused by the unwinding of the double helix.

DNA Polymerases

DNA polymerase can only add to the 3' end, so the 5' end of the primer remains unaltered DNA polymerase III does most of the elongation work, adding nucleotides one by one to the 3' end of the new and growing single strand. DNA polymerase I and RNase H, are responsible for removing the RNA primer after DNA polymerase III has begun its work, replacing it with DNA nucleotides

Replication

DNA replication is a process by which a double-stranded DNA molecule is copied into two, identical DNA molecules DNA replication occurs during the S phase of cell division

Bacterial DNA Replication: Initiation and Unwinding

During initiation, so-called initiator proteins bind to the replication origin, a base-pair sequence of nucleotides known as oriC. This binding triggers events that unwind the DNA double helix into two single-stranded DNA molecules. When the double helix unwinds, replication proceeds along the two single strands at the same time but in opposite directions (i.e., left to right on one strand, and right to left on the other) This forms two replication forks that move along the DNA, replicating as they go

Wobble Mispairings

Example of DNA replication errors Caused by mispairings Either between different but nontautomeric chemical forms of bases (e.g., bases with an extra proton, which can still bind but often with a mismatched nucleotide, such as an A with a G instead of a T) or between "normal" bases that nonetheless bond inappropriately (e.g., again, an A with a G instead of a T) because of a slight shift in position of the nucleotides in space

Tautomeric Shifts

Example of a replication error Both the purine and pyrimidine bases in DNA exist in different chemical forms, or tautomers, in which the protons occupy different positions in the molecule The Watson-Crick model required that the nucleotide bases be in their more common "keto" form Scientists believed that if and when a nucleotide base shifted into its rarer tautomeric form (the "imino" or "enol" form), a likely result would be base-pair mismatching

Leading Strand

In unwound DNA, the single strand that is replicated continuously, in contrast to its partner the lagging strand.

Replication Error --> Mutation

Incorrectly paired nucleotides that still remain following mismatch repair become permanent mutations after the next cell division. This is because once such mistakes are established, the cell no longer recognizes them as errors

Bacterial DNA Replication Stages

Initiation Unwinding Primer Synthesis Elongation

Eukaryotic DNA Replication vs Bacterial DNA Replication: Similarities

Initiation requires a primer, elongation is always in the 5'-to-3' direction, and replication is always continuous along the leading strand and discontinuous along the lagging strand

Eukaryotic DNA Replication vs Bacterial DNA Replication: Differences

One difference is that eukaryotic replication is characterized by many replication origins (often thousands), not just one, and the sequences of the replication origins vary widely among species Eukaryotic replication also utilizes a different set of DNA polymerase enzymes (e.g., DNA polymerase δ and DNA polymerase ε instead of DNA polymerase III). In eukaryotes, the DNA template is compacted by the way it winds around proteins called histones Whereas bacterial chromosomes are circular, eukaryotic chromosomes are linear. During circular DNA replication, the excised primer is readily replaced by nucleotides, leaving no gap in the newly synthesized DNA. In contrast, in linear DNA replication, there is always a small gap left at the very end of the chromosome because of the lack of a 3'-OH group for replacement nucleotides to bind. If there were no way to fill this gap, the DNA molecule would get shorter and shorter with every generation

Bacterial DNA Replication: Primer Synthesis

Primer synthesis marks the beginning of the actual synthesis of the new DNA molecule

Primers

Primers are short stretches of nucleotides (about 10 to 12 bases in length) synthesized by an RNA polymerase enzyme called primase Primers are required because DNA polymerases, the enzymes responsible for the actual addition of nucleotides to the new DNA strand, can only add deoxyribonucleotides to the 3'-OH group of an existing chain and cannot begin synthesis de novo. Primase, on the other hand, can add ribonucleotides de novo. Later, after elongation is complete, the primer is removed and replaced with DNA nucleotides.

Mismatch Repair

Process that corrects mismatched nucleotides in DNA after replication has been completed. Enzymes excise incorrectly paired nucleotides from the newly synthesized strand and use the original nucleotide strand as a template when replacing them. Incorrectly paired nucleotides cause deformities in the secondary structure of the final DNA molecule. During mismatch repair, enzymes recognize and fix these deformities by removing the incorrectly paired nucleotide and replacing it with the correct nucleotide

Repair Enzymes

Repair enzymes recognize structural imperfections between improperly paired nucleotides, cutting out the wrong ones and putting the right ones in their place

Lagging Strand

Replication along it is called discontinuous replication. The double helix has to unwind a bit before the synthesis of another primer can be initiated further up on the lagging strand. Synthesis can then occur from the 3' end of that new primer. Next, the double helix unwinds a bit more, and another spurt of replication proceeds. As a result, replication along the lagging strand can only proceed in short, discontinuous spurts

Strand Slippage

Slipping of the template and newly synthesized strands in replication in which one of the strands loops out from the other and nucleotides are inserted or deleted on the newly synthesized strand A newly synthesized strand loops out a bit, resulting in the addition of an extra nucleotide base The template strand loops out a bit, resulting in the omission, or deletion, of a nucleotide base in the newly synthesized, or primer, strand Most nucleotide insertion and deletion mutations occur in areas of DNA that contain many repeated sequences (also called tandem repeats), and the strand-slippage hypothesis can explain why this was the case When slippage takes place, the presence of nearby duplicate bases stabilizes the slippage so that replication can proceed. During the next round of replication, when the two strands separate, the insertion or deletion on either the template or primer strand, respectively, will be perpetuated as a permanent mutation

DNA Repair

Successful organisms have thus evolved the means to repair their DNA efficiently but not too efficiently, leaving just enough genetic variability for evolution to continue.

Bacterial DNA Replication: Elongation

The addition of nucleotides to the new DNA strand Begins after the primer has been added. Synthesis of the growing strand involves adding nucleotides, one by one, in the exact order specified by the original (template) strand DNA is always synthesized in the 5'-to-3' direction, meaning that nucleotides are added only to the 3' end of the growing strand The 5'-phosphate group of the new nucleotide binds to the 3'-OH group of the last nucleotide of the growing strand

Okazaki Fragments

The fragments of newly synthesized DNA along the lagging strand

Origin of Replication

The location at which a DNA strand begins to unwind into two separate single strands

DNA Ligase

When these enzymes finish, they leave a nick between the section of DNA that was formerly the primer and the elongated section of DNA. Another enzyme called DNA ligase then acts to seal the bond between the two adjacent nucleotides

Pulse Chase Experiments

involved exposing replicating DNA to a short "pulse" of isotope-labeled nucleotides and then varying the length of time that the cells would be exposed to nonlabeled nucleotides. This later period is called the "chase" (Okazaki et al., 1968). The labeled nucleotides were incorporated into growing DNA molecules only during the initial few seconds of the pulse; thereafter, only nonlabeled nucleotides were incorporated during the chase. The scientists then centrifuged the newly synthesized DNA and observed that the shorter chases resulted in most of the radioactivity appearing in "slow" DNA. The sedimentation rate was determined by size: smaller fragments precipitated more slowly than larger fragments because of their lighter weight. As the investigators increased the length of the chases, radioactivity in the "fast" DNA increased with little or no increase of radioactivity in the slow DNA. The researchers correctly interpreted these observations to mean that, with short chases, only very small fragments of DNA were being synthesized along the lagging strand. As the chases increased in length, giving DNA more time to replicate, the lagging strand fragments started integrating into longer, heavier, more rapidly sedimenting DNA strands