Formation of Replication Bubbles

Replication proceeds from a single ori in the circular bacterial chromosome, composed of roughly 6 X 106 bp of DNA. This process is completed in about 30 minutes, a replication rate of 3 X 105 bp/min. The entire mammalian genome replicates in approximately 9 hours, the average period required for formation of a tetraploid genome from a diploid genome in a replicating cell. If a mammalian genome (3 X 109 bp) replicated at the same rate as bacteria (ie, 3 X 105 bp/min) from but a single ori, replication would take over 150

hours! Metazoan organisms get around this problem using two strategies. First, replication is bidirectional. Second, replication proceeds from multiple origins in each chromosome (a total of as many as 100 in humans). Thus, replication occurs in both directions along all of the chromosomes, and both strands are replicated simultaneously. This replication process generates "replication bubbles" (Figure 36-17).

The multiple sites that serve as origins for DNA replication in eukaryotes are poorly defined except in a few animal viruses and in yeast. However, it is clear that initiation is regulated both spatially and temporally, since clusters of adjacent sites initiate replication synchronously. There are suggestions that functional domains of chromatin replicate as intact units, implying that the origins of replication are specifically located with respect to transcription units.

During the replication of DNA, there must be a separation of the two strands to allow each to serve as a template by hydrogen bonding its nucleotide bases to the incoming deoxynucleoside triphosphate. The separation of the DNA double helix is promoted by SSBs, specific protein molecules that stabilize the single-stranded structure as the replication fork progresses. These stabi-

Origin of replication

"Replication bubble"

"Replication bubble"

Unwinding proteins at replication forks

Origin of replication

Directions of replication

Figure 36-17. The generation of "replication bubbles" during the process of DNA synthesis. The bidirectional replication and the proposed positions of unwinding proteins at the replication forks are depicted.

lizing proteins bind cooperatively and stoichiometrically to the single strands without interfering with the abilities of the nucleotides to serve as templates (Figure 36-13). In addition to separating the two strands of the double helix, there must be an unwinding of the molecule (once every 10 nucleotide pairs) to allow strand separation. This must happen in segments, given the time during which DNA replication occurs. There are multiple "swivels" interspersed in the DNA molecules of all organisms. The swivel function is provided by specific enzymes that introduce "nicks" in one strand of the unwinding double helix, thereby allowing the unwinding process to proceed. The nicks are quickly resealed without requiring energy input, because of the formation of a high-energy covalent bond between the nicked phosphodiester backbone and the nicking-sealing enzyme. The nicking-resealing enzymes are called DNA topoisomerases. This process is depicted diagrammati-cally in Figure 36-18 and there compared with the ATP-dependent resealing carried out by the DNA ligases. Topoisomerases are also capable of unwinding su-percoiled DNA. Supercoiled DNA is a higher-ordered structure occurring in circular DNA molecules wrapped around a core, as depicted in Figure 36-19.

There exists in one species of animal viruses (retroviruses) a class of enzymes capable of synthesizing a sin-

Step 1 DNA topoisomerase I = E

Step 2

Step 3

(AMP-Enzyme)

Enzyme (E) -generated -Ö 3' single-strand nick

"Ö Formation of high-H energy bond

Nick repaired

Single-strand nick ' present

Nick repaired

Figure 36-18. Comparison of two types of nick-sealing reactions on DNA. The series of reactions at left is catalyzed by DNA topoisomerase I, that at right by DNA ligase; P = phosphate, R = ribose, A = ademine. (Slightly modified and reproduced, with permission, from Lehninger AL: Biochemistry, 2nd ed. Worth, 1975.)

Figure 36-19. Supercoiling of DNA. A left-handed toroidal (solenoidal) supercoil, at left, will convert to a right-handed interwound supercoil, at right, when the cylindric core is removed. Such a transition is analogous to that which occurs when nucleosomes are disrupted by the high salt extraction of histones from chromatin.

Figure 36-19. Supercoiling of DNA. A left-handed toroidal (solenoidal) supercoil, at left, will convert to a right-handed interwound supercoil, at right, when the cylindric core is removed. Such a transition is analogous to that which occurs when nucleosomes are disrupted by the high salt extraction of histones from chromatin.

gle-stranded and then a double-stranded DNA molecule from a single-stranded RNA template. This polymerase, RNA-dependent DNA polymerase, or "reverse transcriptase," first synthesizes a DNA-RNA hybrid molecule utilizing the RNA genome as a template. A specific nuclease, RNase H, degrades the RNA strand, and the remaining DNA strand in turn serves as a template to form a double-stranded DNA molecule containing the information originally present in the RNA genome of the animal virus.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment