In addition to a local response, the phosphorylated CHK1s generated by a given lesion also contribute to a call for a global, cell-wide response--that response being cell cycle arrest  at the intra-S-phase checkpoint. But phosphorylated CHK1s generated by one or a small number of lesions won't be enough to initiate such a response. Cell cycle arrest can only be initiated by a quantity of phosphorylated CHK1 in the nucleus that can only be reached if there are many lesions or

Fork reversal If a DNA lesion isn't addressed by TLS or repriming in the first 20-30 minutes following RPA-ssDNA detection, fork reversal  will begin to unfold. In  this process, cell proteins rearrange the replication fork into a shape that makes the problematic lesion easier to repair. More precisely, the lesion is removed from the crowded active replisome environment with its clamps, polymerases, etc. and returns it back to the context of dsDNA, where it will be much easie

In the last post, we talked about fork protection , a posture that replication forks take when a lesion stalls a polymerase. Fork protection unfolds in minutes to tens of minutes after the cell detects RPA-dsDNA. Recall that it stabilizes the replisome structure and protects exposed DNA, giving the cell time to deal with the lesion. But fork protection is only the first part of a two-part process called fork stabilization . Fork stabilization also includes fork reversal. How

In the last post, I introduced the cell's two main DNA lesion repair pathways-- base excision repair (BER) and nucleotide excision repair (NER) . Both operate well beyond the replication fork during all of the phases of the cell cycle. But what happens if a lesion escapes BER and NER and finds itself in S phase in front of a moving replication fork? Most DNA lesions get past the CMG helicase but not the DNA polymerase, although some very large lesions can't get past the heli

In addition to DNA polymerase errors, the human genome--even in a perfectly healthy cell--is under constant assault from endogenous  (internal to the cell) and exogenous  (external to the cell, or environmental) chemicals and other agents that alter its chemistry, break its backbone, and distort its helical structure. In this post, we'll first consider what lesions are. We'll see that some are modest (e.g., a small chemical change to the base on a nucleotide) and some are mo

As I mentioned in the last post, DNA polymerases sometimes insert a ribonucleotide (an RNA monomer) into a growing chain instead of a deoxyribonucleotide (a DNA monomer). When that happens, the bases do pair correctly. But the extra small chemical group that differentiates a deoxyribonucleotide from a ribonucleotides can still causes problems like strand breakage. DNA vs RNA . The sugar portion in a ribonucleotide is a ribose whereas in a deoxyribonucleotide it is a deoxyribo

Life can't exist without the high fidelity transmission of genetic information from parent cell to daughter cell. DNA polymerases plays the central role in accomplishing this. DNA polymerases delta and epsilon only make an error (that is, attach a non-complementary nucleotide to a growing chain) once every 100,000 to 1 million nucleotide additions. But this is still too many. Those that arise must be fixed via a back-up system. In addition to polymerase errors, the human ge

As the CMG helicase moves along the double helix separating it into a leading and a lagging strand, it creates what is often referred to as the "winding problem." Let me explain what this is with the help of a human-scale analogy. Consider two ropes wound around each other like a double helix coil. If we attach one end of these two ropes permanently to, say, a table, and then step back and pull the ropes apart to unwind them (in much the same way that CMG helicase unwinds dou

DNA polymerases can only synthesize DNA in the 5' to 3' direction. That is an absolute rule that's determined by the biochemistry of nucleotide addition. Given the antiparallel nature of DNA, a corollary is that DNA polymerases can only synthesize off template DNA that is oppositely oriented in the 3' to 5' direction. Our leading strand exited the MCM2-7 ring of the CMG helicase 3' end first. So the DNA strand that will be synthesized using that strand as a template will be s

An important fact: The two DNA strands in a double helix are oriented in an antiparallel manner. That is, they run in opposite directions like the lanes of a two-lane highway. Recalling chapter ___, scientists describe the orientation of a given single strand of DNA as running either “3’-to-5’” (three prime to five prime) or “5’-to-3’” (five prime to three prime) based on the orientations of the deoxyribose sugars in the DNA backbone. But as a hard and fast rule, DNA polymera

Let's take a closer look at two amazing molecular machines found in the replisome: the CMG helicase and the PCNA sliding clamp . Both are multi-protein complexes arranged as rings that surround DNA. But they have different jobs, different shapes, and interact with the DNA they surround in different ways. Quickly comparing them, the job of the CMG helicase is to propel the replisome forward along double-stranded DNA and at the same time separate that double-stranded DNA into

Our cell of interest is now in S phase and 30,000-50,000 replication origins along its genome have become licensed. The replication origins are licensed based on the presence of two MCM2-7 protein complexes (the licensing factors) lined up head-to-head forming a MCM double hexamer, or MCM-DH. Assembling a functioning replisome starting from an MCM-DH at a replication origin will involve the following four steps: (1) transformation of each of the MCM2-7s into a complete (but s

As we kick off the focus of this book—a deep dive into human genome replication—we’ll see that the process involves many different proteins operating in an extremely well-orchestrated and interconnected manner. Tight regulation of the process is necessary to ensure the genome is replicated once and only once and with extremely high accuracy.     Although the copying of the human genome occurs during S-phase of the cell cycle, the events described in this post--that is, the at

If someone had wanted a book in the 15th century it would have had to have been copied by hand from an existing one. The scribe would have started at page one and proceeded page by page until the last page was, as they say, “in the books.”   A human genome is like a book. So, to copy it, one might imagine the cell would use the same straightforward approach: start at one end of each of the 46 chromosomes and progress to the other. Unfortunately, that wouldn’t work. Human cell

In this post I present a perfect example of ATP-driven protein motor movement. ATP is a frequently employed driver of protein movement in all living cells. The goal here is to absorb a general concept that we'll see several more times as we learn about genome replication. Specifically, today, we'll look at myosin , a protein complex found in skeletal muscle cells. We'll see what, exactly, makes myosin move and how minute movements at the molecular level in muscle cells lead t

Each time a cell divides, it creates a copy of both its genomes for the two daughter cells. It does so with extremely high accuracy (or "fidelity" in molecular biology parlance). Human cells copy their genomes with 99.99999999% accuracy. That equates to one single error per every 100 million nucleotides! Stop and think about that! In this post, we're going to review eight reasons why replicating the human genome is so challenging. Of course, the cell overcomes these challen

A cell is a dynamic place. Enzymes are speeding up chemical reactions. DNA, RNA, and proteins are being synthesized. Molecular motors are carrying organelles around the cell. Enzymes are repairing DNA lesions. The cell membrane is selectively letting specific molecules and ions in and out of the cell. The list goes on. It's a bustling environment! But what is the energy source fueling all this activity? One good answer is "food." The food we eat consists of various kinds of

Two human genomes adding up to 6 billion base pairs are contained in the nucleus of every cell in your body. Different kinds of cells use different genes. That depends on the cell’s job. But every cell contains the whole genome, or every human gene.   One of the cell's challenges is that the genome is very large but its nucleus is very small. If we could stretch out the human genome--in other words line up the 6 billion base pairs end to end--it would stretch nearly 6 feet! Y

We've covered proteins : polymers of amino acid monomers that fold up and perform most of the tasks in the cell. We've also covered DNA : a polymer of nucleotide monomers found in the nucleus that provides the codes needed to make proteins. What we need now is to better understand the relationship between these two kinds of macromolecules. Fortunately, the relationship between the two was clarified in 1958 by James Watson of Watson and Crick fame (discoverers of the struct

The last chapter covered structure: what DNA is. In this one, I move on to function: what DNA does, or, maybe better, how DNA works. I'll start, though, by quickly refreshing some points about structure. Recall that DNA is a polymer made of monomers called nucleotides. There are four different nucleotides that we refer to using their initials: A, T, G, and C. I also mentioned that the double helix form of DNA is really two DNA molecules (polymers) intertwined and connected by